THE METABOLISM OF D-FRUCTOSE
IN AEROBACTER AEROGENES

Thesis for the Degree 9f Ph. D.
MICHIGAN. STATE UNIVERSITY
THOMAS EARL HANSON
1969

   
   

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This is to certify that the

thesis entitled

The Metabolism of D-Fructose

in Aerobacter aerogenes

presented by

THOMAS E. HANSON

has been accepted towards fulfillment
of the requirements for

Ph .D . degree in Biochemistry ..

Major professor

Date September 2, 1969

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ABSTRACT

THE METABOLISM OF D-FRUCTOSE
IN.AEROBACTER AEROGENES

BY

Thomas B. Hanson

A new pathway for the metabolism of D—fructose has been
:found. l2_vitro studies and the analysis of mutants exhib-

iting impaired metabolism of fructose indicate that fructose

is metabolized in Aerobacter aerogenes via the reactions:

 

 

(l) Phosphoenolpyruvate-dependent phosphotransferase

 

D-fructose D-fructose-l-phosphate
+ > +
phosphoenolpyruvate pyruvate

(2) Fructose~l-phosphate kinase

 

Defructosewluphosphate D-fructose~l,6wdiphosphate
+ 9 +
adenosine Slmtriphosphate adenosine 59-diphosphate

D-Fructose is phosphorylated with phosphoenolpyruvate
at carbon atom l by a phosphotransferase system which has
been resolved into four components: enzyme I, HPr and two
components required for enzyme II activity. The enzyme 11

components are a high molecular weight protein and an induc-

ible protein of lower molecular weight, which is proposed to

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Thomas E. Hanson

be the determinant of sugar specificity for a multicomponent
enzyme II system.

Fructose-l-phosphate kinase, an enzyme which catalyzes
the phosphorylation of fructose-l-phosphate with adenosine
SV-triphosphate to yield fructose-l,6-diphosphate, has been
purified and the reaction established. The inducibility of
the enzyme by fructose and its high affinity for its sub-
strates indicate that it has properties consistent with an

in vivo role in the metabolism of fructose. A 6-fructou

 

kinase and a sucrose activity have also been found in fruc-
tose-grown cells. Induction patterns suggest that the 6-
fructokinase is involved primarily in sucrose metabolism
and that fructose-l-phoSphate kinase is not involved in the
metabolism.of sucrose.

Partial purification and characterization of a hexose
phosphatezhexose (H50) phosphotransferase that catalyzes
the formation of fructose-Inphosphate indicated that it
was probably not significant as an in 31!2_source of fruc-
tose-l-phosphate. Establishment of the reactions catalyzed
and kinetic data indicated that the enzyme has a reasonably
high affinity for D-glucose (Km = 0.002 M) and that besides
a variety of hexose phosphotransferase reactions it cata-

lyzes,_at relatively highrates, phosphorylation with phos-
phoramidate and the hydrolysis of the monophosphates of

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Thomas E. Hanson

fructose and glucose. Temperature-dependent modifications
of chromatographic and kinetic properties of the enzyme
'were observed.

Mutants of Aerobacter aerogenes were isolated which
exhibited impaired metabolism of fructose and which lacked
either the inducible component of the enzyme II system
for fructose or fructose-l—phosphate kinase. Characteri~
zation of these mutants indicated that fructose—l—phosphate
kinase and the inducible component of the enzyme II system
are instrumental in the in 3132 metabolism of exogenously
supplied fructose. Also described are the results of
collaborative investigations of mutants lacking 6-fructo—
kinase, fructose diphosphate phosphatase and fructose-6-
phosphate kinase. These investigations establish the
role of 6-fructokinase in sucrose metabolism and the in

vivo relationship between the metabolism of fructose via

 

fructose-l-phosphate and via fructose-é-phosphate. These
investigations have also indicated the relationship of the
fructose-l-phosphate pathway to the gluconeogenic and
glycolytic pathways. The preliminary analysis of a mutant

lacking aldolase is also described.

 

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THE METABOLISM OF D-FRUCTOSE

IN.ABRORACTER AFROGENES

 

By

Thomas Earl Hanson

A THESIS

Submitted to

Michigan State Universitv
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1969

QW—BS
4,2}70

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TABLE OF CONTENTS

Page
A CKNO‘V LEDG DE NTS O O O O O O- 0 O O O O O O O 0 O O O O O O O O 000000 O O O O o 0 O O O O O i 1
LIST OF TABLES. O 0 O O O O O O O O O o 0 O 00000 O O O O O O O O O O O O O O 0 0 O O O O O Viii

LIST OF FIGURES........................................ xi
LIST OF SYMBOLS AND ABBREVIATIONS xv
GENERAL INTRODUCTION ..... ...... ...... 1
SECTION 1 FRUCTOSE-INDUCBD ENZYMES: l. FRUCTOSB-l-

PHOSPHATE KINASE, A NEW ENZYME INSTRUMENTAL
IN THE METABOLISM OF’FRUCTOSE. 2. A SPECIFIC

FRUCTOKINASE. 3. SUCRASE................... 3
1.1 Introduction ............................... 3
1.2 Results .................................... 6
1.2.1 Apparent absence of an FIP aldolase ........ 6
1.2.2 Detection and preliminary characteri—

. zation of fructose-l—phosphate kinase ...... 8
1.2.3 Further purification or FlPK ............... 17
1.2.h Identification of the product of the

FIPK reaction .............................. 27
1.2.5 Detection of befructokinase ................ 31
1.2.6 Detection of sucrose activity in

fructose-grown cells ....................... 35
1.3 Discussion ..... ........................... 37
1.3.1 Establishment of the reaction

catalyzed by FIPK 0000000000000000000000000. 37
1.3.2 The in vivo role of FIPK ................... to

 

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1.3.3 Other investigations of FIPK.4........... hi

1.3.h Supplementary discussion - Review
V of the investigation of mammalian
fructose metabolism...................... uh

l.h Methods.................................. 52
1.h.1 Growth of cells and preparation

of crude extracts ........................ 52
1.h.2 ' Assays ..... .............................. 52
1.h.2.A General techniques for enzymatic assays.. 52
l.h.2.B Enzyme units ..... ........................ 53
l.h.2.C Enzymatic assays.. ........ ...s............ 5h
1.h.2.D Other assays.. ....... .................... 56
1.h.3 Enzyme purification...................... 56

l.h.3.A General enzyme purification techniques... 56
loll—.308 Purification Of FlPK.COOCOOCOOOOOOOOOOOOC 57
l.h.3.C Purification of O-Tructokinase........... 59

l.h.h Preparation and identification of
the prfidUCt 6f.FIPK0.000.000.000000000000 60

1011.05 Reagents0.0000000000000000000000000...... 62
SECTION 2' ISOLATION AND CHARACTERIZATION OF A

HEXOSE PHOSPHATE :HEXOSE (H20) PHOS PHO-
TRANSFERASE OF UNKNOWN FUNCTION. . . . . . . . . . on

2.1 Introduction............................. 6h
2.? Results...000.00.00.00.000DOOOOOOOOOOOOO. 65
2.2.1 Detection of glucose-l-phosphate

dependent formation of fructose-
1‘phasphateooooooooooococoa-0000000000000 65

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2.2.2

2.2.3

2.2.h

2.3

2.11
2.11.1
2.11.2
2.11.2.A
2.h.2.B

2.11.3

2.11.11
2.h.5

SECTION 3

.3.1

3.2
3.2.1

3.2.2

Purification of the H-phosphotrans-
ferase and identification of reactions
catalyzed0000.000000000000COOOOOOOOO0.0...

Temperature-dependent changes in
phosphotransferase prOperties.............

Other properties... ............. ..........
Discussion................................
Methods... ....... .........................
Purification of the H-phosphotransferase..
Assays....................................
Enzymatic assays..........................
Other assays..............................

Attempts to isolate H-phosphotrans-
ferase products...........................

Osmotic shock treatment...................

Reagents 0......00.0.0.0...OOOOOOOOOOOOOOOO

PHOSPHOENOLPYRUVATE-DEPENDENT FORMATION
OF FRUCTOSE-l-PHOSPHATE BY’A FOUR-COMP
PONENT PHOSPHOTRANSFERASE SYSTEM . . . . . . . . . .

Introduction..............................
Results00.0000000000000000.000.000.0000...

UhsucCessful initial attempts to
purify PEP system components..............

A continuous spectrophotometric assay
for a one-step resolution of PEP
system components.........................

Requirements of the assay for the
mannitol PEP system during purifi-

cationC.0.00......OOOOOOOOOOOOOOOOO0.00...

V

Page

68

'86

87
106
115
115
116
116
118

119
120
120

122
122
125

125

126

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3.2.5

3.2.6

3.2.7

3.3
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3.h.2.A
3.h.2.B
3.h.2.C

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Page

Preperties of the fructose PEP system
in crude extracts..........................lu7

Comparative purification of the mannitol

and fructose PEP system - Apparent modi-
fication of the affinity of enzyme II

for fructose...............................169

Requirements of the fructose PEP systems...181

Apparent nonidentity of high Km enzyme
II systems.0....CCOOOCCCC.‘O...............195

Activation by the Km factor and purifi-
cation of Enz I Mtl (gf)...................199

Discu551on OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO208

Possible effects producing an arti-
factual Km factor .........................209

Models for enzyme II activities....,,,.,,,,21h
Other multi-component enzyme 11 systems....220

Supplementary discussion - Relationship
of the fructose enzyme 11 system to
transport00000.OOOOOOOOOOOOOOOOCOOO0......221

thhods ...................................229
Preparation of extracts ...................229
Purification of PEP system components......229
Gel filtration ............................229
Heat treatment of HPr fractions ...........231

Calcium phosphate gel purification
6f Enz IMtl (9f)OOOOOOOOOOOOOOOOOOOO000..231

Assay for PEP-dependent conversion‘
of fructose to fructose-l-phosphate
(fructose assay) ..........................232

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3.h.h Assay for PEP-dependent conversion

of mannitol to mannitol-l-phosphate
(mannitol assay).......................... 23h

3.h.5 Purification of mannitol-l-phosphate
dehydrogenase............................. 23h

3.h.6 Assay for enzyme II for mannose........... 235

SECTION A MUTATIONAL.ANALYSIS OP FRUCTOSE
WTABOLISIVI‘ooooooooooooooooococooooooooooo 237

h.l Introduction ............... ........ ...... 237
h.2 Results and Discussion ........ ....... .... 239
h.2.1 Techniques for mutant isolation... ...... .. 239
h.2.2 Mutants lacking the IOW'Km PEP

system for fructose ...................... 270

u.2.3 Hexose phosphatezhexose (H23) phOSpho—
transferase levels of pleiotropic
mutants 0000000...OOOOOOOOOOOOOOOOOOOOOOOO 278

h.2.h Mutants lacking F1PK...................... 280
h.2.5 Constitutive metabolism of fructose....... 300
h.2.6 Pleiotropic mutants lacking FDPase

or F6PK .................................. 307
h.2.7 A mutant lacking aldolase activity........ 31h
ho3 Methods .................................. 322
h.3.1 Mutant isolation ......................... 322
h.3.2 Analysis of intermediate levels in

mutants .................................. 325
h.3.3 Determination of acid production ......... 326
h.3.h Isolation of a bacteriophage ............. 327
REFERENCES .................... ..... ................. 329

 

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LIST OF TABLES

Page

SECTIONI
1.1 Fractionation for PDP aldolase............. 7
1.2 Induction of FIPK ......... . ....... ........ 10
1.3 Requirements for the aldolaseux-glycero-

phosphate dehydrogenase-linked assay for

FIPKooooooooooooo0.0000000000000000.ooooo.12
l.h Requirements for the pyruvate kinase-

lactate dehydrogenase-linked assay

for FIPK 00.000000000000000...0.00.0.0...O,13
1.5 Purification of FlPK ...................... 21
1.6 Requirements for the aldolaseAX-glycero-

phosphate dehydrogenase assay using 70-

fOld purified FIPK OOOOOOOOOOOOOOOOOOOOOOOO22
1.7 AnalySIS Of FIPK preduCt OOOOOOOOOOOOOOOOOO 30
1.8 Identification of the dephosphorylated

product as fructose ....................... 32
1.9 Requirements for 6-FK ..................... 3h
1.10 Induction patterns of FIPK, 6-FK and

sucrase 0000000....OOOOOOOOOOOOOOOOOOOOOOO. 36
SECTION 2
2.1 Apparent FlP production by crude extracts.. 67
2.2 Ammonium sulfate fractionation for H-

phosphotransferase activities.............. 69
2.3 Complete purification procedure ........... 71

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2.h Requirements of the G1P,F : FlP assay ..... 77
2.5 Requirements of the F1P,G : GP assay ...... 78
2.6 Products formed in H-phosphotransferase

reaCtionSoooooooooooooooooooooooooooo ccccc 80
2.7 Substrate affinities ...................... 82
2.8 Substrate specificity in phosphatase

I‘GBCEIOHS 00000000000000...0000000000000... 814»
2.9 Phosphatase activities - miscellaneous ,,,, 85
SECTION3
3.1 Requirements of the mannitol assay -

CFUde eXtraCtsoooooooooooooooooooooooooooo13LI'
3.2 Requirements of the mannitol assay —

QGI-filtered components 00000000000000.0000135
3.3 Requirements of the mannitol assay -

DEAE cellulose-purified components ........l37
3.h Summary of fructose PEP system

activities in crude extracts.............. ION
3.5 Summary of kinetic parameters of enzyme

II fer fructose0......OOOOOOOCOOOOOOOOOOO.173
3.6 Requirements for the fructose assay

using gel-filtered components............. IBM
3.7 Mannitol inhibition of Enz II Mtl (gf).... 196
3.8 Inhibition of the high Km activity for

fructose in crude extracts ............... 198

3.9 Effect of purification of Enz I Mtl (gf)
on enzyme II activities .................. 200

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SECTIONLL
h.l Enzymatic characterization of mutants-o... 262
h.2 Enzymatic defect in PEP system mutants.... 272
h.3 Enzyme activities in mutants lacking

FIPK and/or 6-m 00......0000.000.000.0000282
h.u Intermediate levels in mutants

lacking FlPK and/or 6-FK ................. 28h
h-S Mutant growth patterns for a random

{salatlon O. ........ OOOOOOOOOOOOOOOOOOO...308
ll.6 Procedures for mutant isolation .......... 323

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LIST OF FIGURES

Page

SECTIONI
1.1 Lineweaver-Burk plots for FlPK ............ l6
1.2 Gel filtration 0f FIPKOOCOOOOOOOOOOOOOOOOO 19
1.3 Effect of aeration on enzyme activities.... 26
l.h Chromatography of FlPK reaction products... 29
SECTION 2
2.1 Coincidence of H-phosphotransferase

activities - gel filtration ............... 73
2.2 Coincidence of H-phosphotransferase

activities - DEAE cellulose ............... 75
2.3 Coincidence of GlP,H20 : G and FIP,G :

GP activities - gel filtration ............ 88
2.h Coincidence of G1P,H20 : G and F1P,G :

GP activities - DEAE cellulose ............ 90
2.5 Temperature-dependent H-phOSphotrans-

ferase forms in DEAE cellulose chroma-

tography.00OOOOOOOOOOOOOOIO0.00.00.00.00... 92
2.6 Effect of temperature on the GlP,H20 :

o and FlP,G : GP activities 95
2.7 Gel filtration in Tris-HCl, pH 7.0 ........ 99
2.8 Phosphotransferases in crude extract

or fI‘UCtose-grom C6115 ooooooooooooooooooolog
9-9 Phesphatase activities EPH 10h

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2.10 Phosphotransferases in crude extract

of glucose-grown cells ........... ........ 105
SECTION 3
3.1 Gel filtration elution profile of a crude

extract of mannitol-grown cells .......... 129
3.2 DEAE cellulose elution profiles of PEP

syStem companents 000000000000000000 00000 01.33
3.3 Effect of ATP on the assay for the

mannitol PEP system ...................... 1M2
3.u Inhibitory effect of Enz II Mtl (gf) ..... 1&5
3.5 FlP formation 23 crude extract concentra-

tion 000000000000000000000000000.000000000 ILL?

3.6 Fructose saturation curves of crude
extracts of fructose-grown cells ......... 153

3.7 Fructose saturation curves for crude
extracts of mannitol-grown cells ......... 155

3.8 Fructose saturation curves for crude
extracts of cells grown on glucose,
glycerol and nutrient broth .............. 157

3.9 Lineweaver-Burk plots for fructose
saturation of crude extracts of mannitol-
and glucose-grown cells .................. 159

3.10 Atypical fructose saturation curve for
crude extract of cells grown on fruc-
tase0000000000000000000000000000000000000161
3.11 Lineweaver-Burk plots for atypical

fructose saturation curve ................ 163
3.12 Non-additivity of activity of crude

extracts of fructose- and glucose-
grm cells 000000000000000000000000000000 168

xii

 

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3.17
3.18

3.19
3.20

3.21
3.22

SECTION E

h.1

h0l0A01
u.l.A.2
h.1.B

Analysis of gel filtration elution

profiles of fructose- and mannitol-

grown cells......................

Fructose saturation curves using gel—

filtered components .......

Lineweaver-Burk plots for high Km
combinations ...

000000000000000000

Lineweaver-Burk plot for low Km
combination..

00000000000000000000

00000000

000000000000000

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Effect of Km factor fraction .............

Rate of FlP formation 22 volume of
enzyme II fraction ............

Time course of FlP production.....

DEAE cellulose chromatography of

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BHZIF-MfBCtOI’ fraCtionooooooooooooo

Rate XE amount of purified Enz I Mtl.....

Rate vs amount of Enz II F using

purifiEd Enz I Mtl ..................

Theoretical effect of product loss

on observed Km...........................

Diagram of the common enzyme 11 base —

specifier protein model..........

00000000

Growth curves of mutants.................

Wild type, PRL-R3..

00.0000000000000000000

Wild type, PRL-R3000000000000000000000000

NMtant QQl7..........

00000000

xiii

000000000000

Page

171

175

177

179
183

188
191

193
aou

206

212

216

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h.l.C MUtant RRIS............................... 252
u-loD Mutant 33336GY.. ........ ..... ............. 252
Lt.l.E Mutant DD31.. .......... . ............. 2511
h.1.F Mutant CCC38 ....... - ..... ....... ...... ..... 25M
h.1.G NMtant Q10.... ...... ...................... 256
h.l.H NMtant 08 .......... .. ..... .. ..... ......... 256
h.1.I NMtant CC7... ....... ...................... 258

h.l.J.l Wild type control for mutant AB........... 260
11.0 10J02 Bflutant 11,8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 260
[4.0 10K Pflutant 012 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 000 0 0 0 0 0 00 0 0 0 0 0 261

h.2 Gel filtration of a crude extract of
fructose-grown cells - QQl7 assay......... 27S

h.3 Current concept of the major pathways
in the metabolism of common carbohy-
drates by'af aerogenes.................... 289

h-u Comparison of acid production in the
presence and absence of mineral
medium“2cocoo-oooooooooooooooooooooooooooo 302

u.5 Saturation curves of acid production...... 305

xiv

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LIST OF SYMBOLS AND ABBREVIATIONS

All sugars are the D isomer unless noted otherwise.

ATP
DEAE
DPN

DPNH

EDTA

EMB agar
enzyme 1
enzyme 11

HPr

Enz I F (gf)
Enz II F (gf)
HPr F (gf)

Enz I Mtl (gf)

EDP

PDPase

adenosine 5'-triphosphate
diethylaminoethyl-
nicotinamide adenine dinucleotide

reduced nicotinamide adenine
dinucleotide

ethylenediaminetetraacetate
eosine methylene blue agar

generic terms for components of
the PEP system as defined in
Section 3.1

terms designating fractions
(containing components of PEP
systems) obtained by gel fil-
tration of crude extracts of
fructose-grown cells (E), or
mannitol-grown cells (Mtl).
(gf) refers to gel filtration;
other purification steps and
the concentration of fractions
are indicated by other symbols.
(See Section 3.u.2.A.)

D-fructose
fructose-1,6-diphOSphate

fructose-1,6-diphosphate phosphatase

XV

 

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FlP
F6P
FIPK
F6PK
FlP,G : GP
and similar
terms
6-FK
G
GlP
G6?
Gy
He:- 1
H-phosphotrans-

ferase

Km factor

M

mineral
medium-2

Mtl
NB

O.D.

fructose-l-phosphate
fructose-6—phosphate
fructose-l-phosphate kinase

fructose-6-phosphate kinase
(6-phosphofructokinase)

a type of format used to describe
reactions and assays in Section 2
(See Section 2.2 and Section
gouogvo)

6-fructokinase

D-glucose

glucose-l-phosphate
glucose-6-phosphate

glycerol

apparent heat of activation

the hexose phosphatezhexose (H20)
phosphotransferase described in

Section 2

the low molecular weight inducible
component of enzyme II for fructose
(See Section 3.2.5.)

D-mannose

a particular type of mineral ,
medium (See Section h.3.2.)

mannitol
nutrient broth (See Section 1.h.l.)

optical density

xvi

PEP

PEP system

Pi

Stl
Tris

TPN
TPNH

phosphoenolpyruvate

phOSphoenolpyruvate-dependent
phosphotransferase system

inorganic phosphate

sorbitol

2-amino-2-(hydroxymethyl)-1,3-
propanediole

nicotinamide adenine trinucleotide

reduced nicotinamide adenine
trinucleotide

xvii

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GENERAL INTRODUCTION

The objective of this work was to find a complete
pathway for fructose metabolism in Aerobacter aerogenes.
The material in this thesis is presented approximately
in chronological order. This is done in order to pre-
serve the continuity of the ideas which directed the
work. It is also necessary due to the rapid advances
which have been made in areas directly related to, or
identical with, most of the topics considered. Section
1 presents the isolation of the more easily detected
enzymes involved in fructose metabolism - fructose-l-
phosphate kinase, 6—fructokinase and a sucrase activity.
Section 2 describes the investigation of a hexose phos—
phate:hexose (H20) phosphotransferase with some unique
properties. This enzyme is included because though it
did not prove to be of significance in fructose metabolism
it was nevertheless necessary to eliminate such a
possibility. Section 3 describes the solution to the
most resistant problem in this work - the origin of
fructose-l-phosphate in fructose metabolism. The phos-
phoenolpyruvate-dependent phosphotransferase which
catalyzes the formation of fructose-l-phosphate is
basically another example of the system discovered by
S. Roseman and his collaborators. The establishment of

this system has had a great impact recently on the once

 

    
 
  

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more or less separate fields of bacterial carbohydrate
metabolism and bacterial carbohydrate transport. Our
work on this system derives significance primarily from
the fact that it has produced for consideration another
protein component in this already complex system. Sec-
tion A describes the analysis of mutants lacking enzymes
in fructose metabolism and some of the important enzymes
in glycolysis and gluconeogenesis. In this section an
attempt is made to produce a consistent picture of fruc-
tose metabolism. A diagram summarizing the current
ideas on fructose metabolism and related areas is given

in Figure h.3.

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SECTION 1. FRUCTOSE-INDUCED ENZYMES: 1. FRUCTOSE-l—
PHOSPHATE KINASE, A NEW ENZYME INSTRUMENTAL IN THE
METABOLISM or FRUCTOSE. 2. A SPECIFIC
FRUCTOKINASE. 3. SUCRASE

SUMMARY} An enzyme which catalyzes the phosphorylation
of fructose-l-phosphate with ATP to yield fructose-1,6-
diphosphate has been purified and the reaction estab-
lished. The inducibility of the enzyme by fructose and
its high affinity for its substrates indicate that it

has properties consistent with an in vivo role in metabo-

 

lism. It is suggested that the reaction is a step in a

new pathway for fructose metabolism. .A 6-fructokinase

and a sucrase activity have also been found in fructose-
grown cells. Induction patterns suggest that the 6-fructo-
kinase is involved primarily in sucrose metabolism.rather
than in the metabolism of exogenously supplied fructose

and that fructose-l-phosphate kinase is not involved in

the metabolism of sucrose.

_l.1. INTRODUCTION
Interest in fructose metabolism in.Aerobacter aerogenos
arose from three observations made in the course of inves-
tigations of hexose metabolism by M.‘Y. Kamel and R. L.
Anderson. First, from oxygen consumption data (M.‘Y. Kamel,

unpublished), and growth curves (Section h), it was evident

3

 

 

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that wild type A, aerogenes, strain PRL~R3, after a

short lag period of 5 to 10 minutes metabolises
fructose at rates comparable to those for other common
hexoses and hexitois. The short lag in metabolism
suggested that the major pathway of fructose metabolism
‘was dependent on an inducible enzyme(s).

Second, preliminary experiments had suggested that
A. aerogenes did not have a fructokinase‘which catalyzed
the formation of F6P (l, 2). It was generally assumed
at the time that the common pathway for fructose metabo-
lism‘was via kinases producing F6P, particularly non-
specific hexokinases such as those in yeast (3). On
the basis of preliminary information, it was believed
that in Escherichig 921; fructose was metabolized via
a hexokinase producing F6? (h. 5).

The third factor, which directly initiated this
investigation'was the isolation by Kamel and.Anderson
of a constitutive hexose phosphate, acyl phosphate:
hexose phosphotransferase from a. aerogenes (6). When
the purified phosphotransferase was used to phospho-
rylate fructose the product was found to contain
fructose-l-phosphate as well as fructose-6-phosphate.
Although it‘was later shown that the production of FlP
by this phosphotransferase probably is not functional
in fructose metabolism, the Km for fructose (0.3 M)

(6) being too high, a preliminary interpretation of the

 

 

  
 

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results was that perhaps fructose was metabolized via
FlP and that there might exist a pathway similar to
that in mammalian liver. This approach was also rein-
forced by the prediction of Liss, Horwitz and Kaplan
(7) that in some unknown way fructose metabolism.in,A.
aerogenes occurs via FlP. The prediction was based on
an ingenious but very indirect rationalization of the
induction pattern of mannitol-l-phosphate dehydrogenase
as described in Section h.2.h.

As presented in the discussion (Section 1.3.h) the
only previously established pathway for the degradation
of FlP is that which functions in mammalian liver. In
this pathway FlP is cleaved by a special aldolase'which
is active with FIP as well as FDP. Thus the first step
in the present investigation was to examine extracts of
A. aerogenes for an enzyme which cleaved FlP. Such an
enzyme could not be detected. Instead, a new enzyme,
fructose-l-phosphate kinase (FlPK), was found. In this
section the preliminary purification and characterization
of this enzyme is described and it is established that
the reaction catalyzed is the phosphorylation of FlP in
the 6 position. Also described is the detection in
fructose-grown cells of a 6-fructokinase and an apparent
sucrase activity.

Portions of this section are covered in references

2 and 8.

    
  

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1.2. RESULTS

1.2.1. APPARENT ABSENCE OF AN FlP ALDOLASE. Assays of
crude extracts of‘A, aerogenes grown on glucose and
fructose with both ancx-glycerolphOSphate dehydrogenase-
linked assay and a hydrazine-dependent assay for triose
phosphate formation gave no activity with FlP as the
substrate. An FDP aldolase activity (specific activity,
0.30 pmoles DPN/min/mg protein) was detectable in both
glucose- and fructose-grown cells and had a Km of less
than 6.6 x 10-3 M. However the sensitivity of any

assay'which measures DPNH oxidation is, for crude extracts

of.£. aerogenes, limited to activities greater than about

 

0.01‘pmoles DPN/min/mg protein due to the activity of
DPNH oxidase. The hydrazine assay (9) in whichsa com-
plex absorbing at 2&0 nm is formed from triose phosphates
is also of limited sensitivity since the absorption of
crude extracts at 2h0 nm is of such a magnitude that
relatively low concentrations of extracts produce
absorbancies above the determinable range.

Ammonium sulfate fractionation for the FDP aldolase
resulted in fractions much reduced in DPNH oxidase
activity. However none of the fractions catalyzed
detectable FlP aldolase activity (Table 1.1). Thus, at
least under the conditions used in these experiments,
the FD? aldolase does not cleave FlP appreciably and there
does not appear to be an appreciable level of FlP aldolase

activity catalyzed by a more specific enzyme.

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8

1.2.2. DETECTION.AND PRELIMINARY CHARACTERIZATION OF
FRUCTOSE-l-PHDSPHATB KINASE. By an inverse analogy
the existence of fructose-é-phosphate kinase which
catalyzes the phosphorylation of F6P in the 1 position
suggested that an enzyme might exist which catalyzed
the phosphorylation of F1? in the 6 position —— a
fructose-l-phosphate kinase. As presented in the
discussion the existence of such an enzyme has been
proposed previously but has never been established.
In order to detect an FlPK, crude extracts were tested
using an assay involving the reaction sequence:
(1) FlPK
FlP + ATP ——> FDP + ADP
(2) Aldolase
FDP e=efiDihydroxyacetone phosphate + glyceral-
dehyde-3-phosphate
(3) Triosephosphate isomerase
Glyceraldehyde-3-phosphate §=e=Dihydroxyacetone
phosphate
(h) <belycerol phosphate dehydrogenase
Dihydroxyacetone phosphate + DPNH —,.=‘~o<-Glycerol
phosphate + DPN+
A fructose-l-phosphate kinase (FlPK) activity of about
0.3 pmole DPN/min/mg protein was detected in extracts
0f fructose-grown cells. The assay was linear with

reSpect to enzyme concentration and to time provided

9
that enough aldolase andcx-glycerolphosphate dehydro-

genase was present. The activity was about 15% higher
at pH 7.5 than at pH 7.0 or 8.0. Testing extracts of
cells grown on various substrates (Table 1.2) indicated
that the activity was fully induced only by fructose.
The presence of other enzymes in crude extracts makes it
impossible to establish that low, uninduced levels of
apparent FlPK activity, are actually catalyzed by FlPK,
‘without purification of the enzyme responsible for such
activities. Phosphatases could catalyze the formation
from PI? of free fructose which could then be converted
to FDP by a 6-fructokinase (see below) and F6PK. The
presence of contaminating free fructose in the PIP could
also result in such an activity. It is possible that
some of the low levels of FIPK in Table 1.2 are due to
artifactual activities, particularly that in the extract
of glucose-grown cells, which was not detected in other
experiments (Tables 1.10 and h.3). In fact all of the
assays available for FlPK are of such a type that even a
high activity detected in relatively unpurified fractions
can easily be attributed to other enzymes. In the past
(see Discussion) the possible presence of F1? aldolase,
in particular, has invalidated results. Thus it was
necessary to purify the enzyme responsible for the FlPK
activity in order to establish the existence of the

reaction as preposed.

10

Table 1.2. INDUCTION OF FlPK

Growth Substrate Specific Activity
(pmoles DPN/

min/mg protein)

Fructose 0.28
L-Sorbose 0.0a
Sorbitol 0.0a
Glucose 0.03
i-Inositol 0.01
Mannose 0.01
Mannitol 0.00

Nutrient Broth 0.00

11
A preliminary purification, involving fractionation

with protamine sulfate, ammonium sulfate and a heat treat-
ment, similar to that shown in the first steps given in Table
Table 1.5.gave a five-fold purified preparation of FlPK.
This was tested for the requirements of the reaction as
assayed with the aldolase-qaglycerolphosphate dehydrogenase-
linked assay and the pyruvate kinase-lactate dehydrogenase-
linked assay (Tables 1.3 and l.h). The<X-glycerolphosphate
dehydrogenase preparation used contained triose phosphate
isomerase so that both of the cleavage products are re-
active in the assay. The lactate dehydrogenase preparation
used was relatively impure making interpretation of resi-
dual activities difficult (see Methods, Section l.h.2.C).
Both assays required all of the expected components for

the reaction proposed. USing the aldolase-ohglycerolphos-
phate dehydrogenase-linked assay, there was a small amount
of activity in the absence of FlPK which was due to P1P
cleavage by muscle aldolase. There was also some activity
in the presence of F6P, about half of which, as suggested by
its.ATP requirement, was catalyzed by contaminating FéPK.
The activity'with F6P which does not require ATP was
probably due to the F6P reductase reaction catalyzed

by contaminating mannitol-l-phosphate dehydrogenase (7).

The F6? activity was additive with respect-to the PIP
activity, indicating separate enzymes and thus eliminating
the possibility that the FlPK activity was due to phospho-
rylation of FlP catalyzed by F6PK. Since the activity

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Table 1.3. REQUIREMENTS FOR THE ALDOLASBixGLYCEROPHCS~
PHATE DEHYDROGENASE-LINKED ASSAY FOR FlPK. The assay
(Methods) contained 1.38 pg protein of FlPK purified
(five-fold) through the heat treatment step.

Reaction Mixture Rate

(pmoles DPN/
min/mg protein)

Complete 1.3h
" minus ATP 0.09
" minus MgCl2 0.02
" minus aldolase 0.02
" minus abglycerophosphate

dehydrogenase-

triose phosphate

isomerase 0.01
" minus FlP 0.00
" minus FlPK fraction 0.09
" minus FlP, plus fructose

(0.0066 M) 0.00

I

" minus FlP, plus F6P (0.0066 M) 0.30
" minus PIP, plus P6P (0 0066 M),

minus ATP 0.16
" plus F6P (0.0066 M) 1.66

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13
Table l.h. REQUIREMENTS FOR THE PYRUVATE KINASE-LACTATE
DEHYDROGENASE-LINKED ASSAY FOR FIPK. The assay (Methods)

contained 2.76 pg protein of the fraction described in

Table 1.2
Reaction Mixture Rate
(pmoles DPN/
min/mg protein)
Complete 1.06
" minus ATP 0.08
" minus lactate dehydrogenase-
pyruvate kinase 0.03
" minus FlPK fraction 0.09
" minus PIP 0.11
" minus PIP plus fructose
(0.0066 M) 0.13
" minus FlP plus glucose
(0.0066 M) 0.2a
" minus FlP plus glucose
(0.0066 M),minus ATP 0.00
" minus FlP plus'F6P
(0.0066 M) 0.6M
" minus FlP plus F6P
(0.0066 M),minus ATP 0.13
" plus F6P (0.0066 M) 1.52
" minus FIP plus DLFgl ceral-
dehyde (0.012 M 0.11
" minus FlP plus DL-glyceral—
dehyde (0.12 M),minus
ATP 0.01

111
with F6P‘was lower than that with FlP it was not possible
for the FlP activity to be due to a conversion of F1? to
F6? via a mutase-type reaction. F6P formation from PIP
was also found not to be catalyzed by crude extracts of
glucose- and fructose-grown cells as determined with a
phosphoglucose isomerase-91ucose-6-phosphate dehydro-
genase-linked assayu [Recently, a very low level of FlP
mutase activity has been found to be a non-specific
reaction of phosphoglucomutase (10);] Using the pyruvate
kinase-lactate dehydrogenase-linked assay (Table l.h) a
higher rate was observed in the presence of F6P. The
reason for this is not known. Mast of the activity was
ATP-dependent and it was approximately additive with the
PIP activity. There is also some glucokinase and fructo-
kinase activity in the five-fold purified preparation.
The absence of appreciable quantities of glyceraldehyde
kinase is evidence that the activity is not due to an
P1P aldolase for which.ATP is required for the phospho-
rylation of D- or L-glyceraldehyde. As seen in Figure
1.1 the Km of HM for‘FlP is about 3 x 10-11 M and that
for.ATP is about 8 x lO'u M, both of which are well‘with-
in the physiological range. There was an inhibition at
high non-physiological levels of.ATP.

"1 (L
(

 

15

Figure 1.1. LINEWEAVER-BURK PLOTS FOR FIPK. Assays:
(1) The ATP concentration was varied, keeping the
MgC12 concentration twice that of.ATP. The FlP con-
centration.ums 0.0066 M (D). (2) The P1P concentra-
tion was varied using 0.0066 M.ATP and 0.012 M MgC12
(o). The PlPK fraction used the same as that
described in Table 1.3. The same amount was used in

both sets of assays.

 

Figure 1.1.

16

 

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17

1.2.3. FURTHER PURIFICATION OF FlPK. FlPK activity was
lost in Sephadex 0100 chromatography using water as the
eluant. However 100% recovery of activity was obtained
when heat treated ammonium sulfate fractions were eluted
from Sephadex 0100 or Bio-Gel P150 with ammonium sulfate
(0.2 M, pH 5.6) or sodium phosphate (0.02-0.05 M, pH 6.5
or 7.5). A typical elution profile is given in Figure
1.2.A. In Figure 1.2.B is shown an atypical elution
profile in which a small peak was found in front of the
major peakfi This small peak was found in four out of
about thirty experiments, including two in which Sephadex
Gi00 was eluted with 0.02 M sodium phosphate, pH 7.5,
which'was the standard method. In one of the cases an
immediate repetition of the experiment using an identical
aliquot of the same sample preparation on the same column
failed to reproduce the small peak.

An attempt was made to purify the gel-filtered pre-
paration by ion-exchange chromatography. The variations
tried are described in Methods, Section l.u.3.B. In all
cases there was an extensive loss of activity or else the
activity occurred in the region of a particular major pro-
tein peak. In the most successful experiment (Methods,
Section l.h.3.C) enough resolution from.the major protein
Peak*was obtained using DEAE cellulose to give a two-fold
Purification, resulting in a final purification of hO-fdld.

The same procedure resulted in an extensive loss of

18

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Figure 1.2.

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activity when scaled up for a larger volume. This was be-
cause a large column of DEAE cellulose with about the same
proportions as a much smaller column allows a relatively
lower flow rate resulting in retention of the enzyme on the
column for a greater length time so that if the enzyme is
unstable in contact with DEAE cellulose,a greater loss of
activity'results.

Calcium ph03phate gel treatment of gel-filtered prepara-
tions gave a final purification of about 70-fold with con-
sistent recovery of about 50 percent for the step, the loss
being due to discarded fractions. A summary of a complete
purification is given in Table 1.5. In Table 1.6 are listed
the assay properties of the preparation resulting from this
particular purification. The 70-fold purified preparation
was completely lacking in activity toward F6P. The prepara-
tion gave no activity with 0.066 M fructose using the aldo-
lase-«x-glycerophosphate dehydrogenase-linked assay. .Activi-
ity was also not observed in a 6-fructokinase assay for F6P
formation which was coupled with phosphoglucose isomerase and
glucose-6-phosphate dehydrogenase. These results indicate
that FlPK has no nonspecific reactivity toward fructose.

Similar highly purified preparations gave negligible
(less than 1%) activity when tested for ability to phos-
phorylate the possible metabolic analogs of F1? - gluco-
nate, 2§keto-3-deoxy-gluconate, 2-ketogluconate and

5-ketogluconate - at 0.066 M using the pyruvate

 

 

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23
kinase-lactate dehydrogenase-linked assay. None of these
compounds inhibited FlPK activity when tested at 0.066 M
in assays containing a half-saturating concentration of
FlP. However,in an FlPK fraction which was purified only
up through Sephadex 0100 chromatography, a detectable
level (5% of the FlPK activity) of an apparent kinase
activity for 2-keto-3-deoxygluconate was found using the
pyruvate kinase-lactic dehydrogenase-linked assay.

The calcium phosphate gel-purified FlPK was used for
end-point assays for FlPK (Section 3.h.3). For these
assays it was purified up through the gel filtration stage,
stored at -2023 (for up to one year without appreciable
loss of activity) and purified with calcium phosphate gel
immediately before use. In some such preparations low
levels of activity for F6P'were still present. This did
not interfere in the discrimination of F1? from F6P due
to the difference in rates. However there were also
small amounts of glucokinase and phosphoglucose isomerase
in these preparations and in the presence of glucose the
interference due to accumulation of F6? was too extensive
to obtain useful assays. Purification with a pH treatment
removed this interfering activity. Purification of FlPK
from an F6PK negative mutant is a more effective solution
for this problem (ll, 12)

In the course of purifying FlPK it was noted that the

specific activity in crude extracts varied up to five-fold

 

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(0.50-0.10 pmole DPN/min/mg protein). It appears that the
degree of aeration is at least partially responsible for
this. In the experiment illustrated in Figure l.u cells
were grown under conditions varying from highly aerobic

to anaerobic in the presence of 0.5% fructose. The final

O'D°600 in the stationary phase was plotted X§.th€ rela-
tive activities of FlPK and aldolase as well as a 6-fructo-
kinase (Section 1.1.5) and a phosphotransferase (Section 2).
Since sugar was the limiting component in the medium the
final O.D.600 is proportional to the cell yield which is in
turn a rough index of the amount of aeration. As can be
seen, all of the activities drop with increasing aeration,
with FlPK and the 6-fruct0kinase paralleling aldolase.

This is probably an example of the Pasteur effect which is
apparently related to the fact that the low energy yield
obtained under anaerobic conditions can be compensated

for by'a higher rate of glycolysis. This effect was also
observed for glucokinase in other experiments. As deter-
mfned.by final O'D'600’ test tube cultures (final O’D'600 =
0.7) and 500 m1 Fernbach cultures (final O.D.600 = 0.8) are
smut—anaerobic since final O°D°6 -va1ues 0f 2.0-2.3 are

00
obtained with forced aeration.

{r L

{ma

.3—

'c-u

 

l
12%“ .

25

Figure 1.3. EFFECT OF AERATION ON ENZYME.ACTIVITIES.

Aldolase (D), FlPK (O), 6-FK (A), hexose
phosphate:hexose (H20) phosphotransferase (O)

assayed for glucose phosphate production from FlP

and glucose (see Section 2).

Assays:

Final O.D.600 and

growth conditions: 0.3M —— airtight Fernbach flask

with shaking; 0.76 —— cotton-plugged Fernbach flask,
500 m1 culture, with shaking; 1.17 —— cotton-plugged
Fernbach flask, 250 ml culture, with shaking; 2.00 ——

forced aeration (Fermicell Fermentor).

F‘.“‘
‘0‘“
.

LATIVE SPECIFIC ACTIVITY (m)

H 1‘1

H

1')
L)

e

n

k

26

Figure 1.3.

 

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100 —— ,/// __

 

 

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50

RELATIVE SPECIFIC ACTIVITY (%)

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27
1.2.h. IDENTIFICATION OF THE PRODUCT OF THE FlPK REACTION.
For reasons given in the discussion an identification of
the product of the FlPK reaction is necessary even though
it is a common intermediate. Specifically, it is necessary
to establish that the sugar moiety of the product is fruc-
tose rather than one of the three other ketohexoses ——
sorbose, tagatose and psicose. As described in Methods,
the product was prepared with a reaction mixture containing

hO-fold purified FlPK and limiting ATP. The pH was main-

‘ tained at 7.5 by titration of the acid produced. The re-

action mixture was chromatographed on a Dowex-l—borate
column employing gradient elution. The peaks in the elu-
tion profile (Figure l.h) were identified by their corre—
spondence with known samples of FIP, ADP, FDP and ATP.

The chromatography system is not expected to discriminate
among different ketohexose diphosphates though it does
separate the common hexose monophosphates. The stoichi-
ometry for the reaction given in Table 1.7, is in fairly
900d accordance with the reaction proposed except that
there is an excess of ADP formed. This may be due to ATP-
ase activity in the FlPK preparation. The material in

the FIDP fraction served as a substrate for rabbit muscle
aldolase. Equivalent amounts (as determined by net change
in O.D.3uo) of the unknown and a known FDP sample gave
SupeI‘imposable plots of O.D. E§ time, a Situation

3’40
which vvould be possible only if the Vmax and Km values

28

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for the two components were identical. The ratio of bound
phosphate to aldolase-reactive material was 2.0. Together
these two facts strongly indicate that there is no keto-
hexose diphosphate present other than fructose diphosphate.
The pooled product‘was then dephosphorylated with acid
phosphatase and the sugar moiety was identified as fructose
(Table 1.8) by paper chromatography, the complete spectra
of the product of the cystein-H28q4reaction, and the
time required in the cystein-H2Sq+reaction for half-
maximal development of O.D. at u12 nm. This half-maximal
value was found to be independent of sugar concentration
(indicating a first order reaction with respect to sugar)
and therefore a useful, and in fact the best, index for

identification of the four possible ketohexoses.

1.2.5. DETECTION OF 6-FRUCTOKINASB. As initially sug-
gested by the presence of a low fructokinase activity
which‘was found in an FlPK preparation (Table l.h) there
is a 6-fructokinase (6-FKJin A. aerogenes. Assays of
crude extracts of fructose-grown cells with a phospho-
glucose isomerase-glucose-6-phosphate dehydrogenase-linked
assay indicated the presence of a kinase which required
ATP and which produced F6? with a specific activity in the
crude extract of about 0.01 pmoles TPNH/min/mg protein.

In glucose-grown cells the specific activity was one-third
or less than that in fructose-grown cells. In a crude ex-

tract of fructose-grown cells the activity had a Km for

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33

fructose of less than 6.6 x 10'3 M. The activity was
purified from an extract of fructose-grown cells using a
protamine sulfate treatment, 0 to 30% ammonium sulfate
fractionation, and chromatography on Sephadex (3100,. to
give a final purification of 35-fold. Characterization

of the purified fraction (Table 1.9) indicated that there
was an additive glucokinase activity and a lack of activity
on a number of other sugars. The purified fraction was
inactive in an assay for FlP formation which was coupled
with FlPK, aldolase and o<¥glycerophosphate dehydrogenase,
indicating that a mixture of FlP and FéP was not formed.

Recently, N. E. Kelkar, of our laboratory, in reinvesti-

gating this enzyme (13), has confirmed the high specificity
and found a Km for fructose of 2 x 10")4 N1. However

using a 6-F‘K negative mutant (mutant 114, see Section 14.2.3)
he found (unpublished) that the apparent basal level of
this activity present in glucose-grown cells is due, not
to the specific 6-FK, but to a low level of a kinase
which is apparently constitutive. This kinase is specific
for fructose (Km is about 2 x lomLL M) and mannose and
pI'Oduces the 6-phosphates only, FlP not being formed. A
metabolic role for this enzyme has not yet been found.

One possibility is that the activity represents the .un-

induced level of an inducible enzyme.

 

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Table 1.9. REQUIREMENTS FOR 6-FK. The gel filtration-

purified fraction was used.

1. Phosphoglucose isomerase-glucose-6-phosphate-dehydro-

genase-linked assay.

 

Reaction Mixture Rate
(pmoles TPNH/
min/mg protein)

 

Tinnplete 0.33
" minus ATP 0.00
" minus MgCl2 0.00
" minus fructose 0.00

minus fructose plus
glucose (0.0066 M) 0.51

" plus glucose (0.0066 M)' 0.83

 

2. P3rruvatekinase - lactate dehydrogenase-linked assay.

 

Reaction Mixture Rate
(pmoles TPNH/
min/mg protein)

 

Complete 0.37
" minus ATP 0.06
" minus fructose 0.06

minus fructose plus

glucose (0.0066 M) 0.58
" plus glucose (0.0066 M) 0.79
'7 minus fructose plus other
sugars” 0.08
or less

 

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35
1.2.6. DETECTION OF SUCRASE.ACTIVITY IN FRUCTOSE-GROWN
CELLS. In the course of examining the possibility that
fructose metabolism is initiated by the formation of
sucrose which then in some way yields FlP (no such path-
way was found), an activity causing TPN reduction in ex-
tracts of fructose-grown cells in the presence of.ATP
and sucrose was detected. In the absence of ATP and/or
TPN, product accumulation occurred, indicating the exis-
tence of an apparent sucrase activity which Was coupled
to TPN reduction via glucokinase, 6-FK, phosphoglucose
isomerase and G6? dehydrogenase, which were present in
the extracts. The glucose released in the reaction
could be assayed using glucose oxidase (the Glucostat
reagent). (It was found that some yeast hexokinase pre-
parations obtained at the time contained high levels of
sucrase making them unsuitable for a hexokinase-linked
assay.) Table 1.10 gives the induction patterns of the
sucrase activity, 6-FK and FlPK. Also given are assays
for cells grown on mixtures of fructose and glucose.
Whether or not the increase in sucrase levels observed

is significant is not known and has not been examined

further.

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37
1.3. DISCUSSION

1.3.1. ESTABLISHMENT OF THE REACTION CATALYZED BY PIPK.
As Inentioned (Section 1.1), there is a pathway in mammalian
liver for the metabolism of fructose involving the partici-
pation of an FlP aldolase. A brief review of the investiga—
tior1 of this pathway is given in Section 1.3.0. Arising
frorn some of the work which ultimately resulted in the
establishment of the PIP aldolase pathway was a suggestion
nmcie by Slein, Cori and Cori (15) that there is an FlPK in
mus<:le. However the existence of FlPK in muscle has never
beer1 substantiated and there has been controversy in the
literature as to the existence of this enzyme (see Section
1.3.24). Thus it was especially important in the present
hunestigation to definitely establish the existence of FlPK
bycietermining that the activity was not due to an artifact
involxdng an PIP aldolase or some other artifactual system.
Distinction from an artifact involving P1P aldolase was
shovnu by, among other things, the fact that with purified
Preparations both ATP and FDP aldolase are necessary for
the production of dihydroxyacetone phosphate from FlP. Al-
though glyceraldehyde kinase, a component involved in the
PIP aldolase pathway, is present in crude extracts, the
apparent absence of an FlP aldolase in crude extracts
indicates that there is probably no pathway in A. aerogenes
like that in liver.

FIP could conceivably be epimerized to the l-phosphate

of either L-sorbose, D-tagatose or D-psicose. Since

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38

L-sorbose-l,6-diphosphate and D-tagatose-l,6-diphosphate
are‘known to be substrates for muscle aldolase (9, 16)
(though the Km and Vmax values are different from those
for IFDP) an epimerase plus a ketose-l—phosphate kinase
migrrt give an apparent FlPK reaction. This possibility
was 'eliminated by the identification as fructose of the
sugar moiety of the ketohexose diphosphate which was
fornued. The specificity of F1? has been checked with
respect to only the more common substrates. The enzyme
may luave activity toward other ketohexose-l-phosphates.
The lack of full induction of FlPK by L-sorbose suggests
that. it is not involved in L-sorbose metabolism.
NbRorie and Novelli (17, 18) have suggested that
D-glt1curonate is metabolized in.A, aerogenes via the
pathmvayw
(1) 'Uronic isomerase

ID-Glucuronate ;==§ D~Fructuronate

(2) ‘Unknown kinase

D-Fructuronate + ATP ——A~D-Fructuronate-l-phosphate
+ ADP

(3) 'Uhknown aldolase

D~Fructuronate-l—phosphate ——A~Dihydroxyacetone

phosphate + Tartronic Semialdehyde.

 

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39

The uronic isomerase was induced by growth on glucuronate.
The kinase and aldolase activities were constitutive and
it “mas proposed that they were catalyzed by the glycolytic
enzynnes, F6PK and FDP aldolase. Fructuronate was proposed
to be: an analog for F6P and fructuronate-l-phosphate, an
analc:g for FDP. The inactivity of FlPK toward 2—keto-
gluccxnate, a possible analog of FlP by a similar rationale,
as well as toward gluconate and other related compounds,
indi<:ates that the FlPK reaction is not catalyzed by an
unkncnwn enzyme which might be involved in gluconate
metal>olism and that gluconate is not metabolized via

FIFK, Growth on gluconate does not induce FlPK (N. E.
Kelker, unpublished results). In view of the control
prOperties of F6PK in A. W (see below) it would
be sxrrprising if fructuronate-l-phosphate could serve
effecrtively 19 Milo as a substrate for this enzyme. The
actixrities reported by MCRorie and Novelli (17) for both
the pfliosphorylation and cleavage were much lower (0.03
andl3.0008 umoles/min/mg protein respectively) than that
Of the isomerase (0.31 pmoles/min/mg protein). Some

doubt has been expressed as to the significance of this
pathway-(19). An inducible uronic isomerase initiates

a well established, but different, pathway in g, 99;; for

glucuronic acid metabolism (20).

 

 

 

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1.3.2. THE IN m ROLE OF FlPK. Thus FlPK appears to
be a new enzyme, catalyzing the phosphorylation of HP

as described, and with no apparent possibility of duality
or ambiguity of its catalytic function except that per-
haps other less common ketohexose-l-phosphates could
conceivably be substrates. Recently these conclusions
were further substantiated by a 3lS-fold purification of
the enzyme (11, 12). The inducibility, substrate specifi-
city, and affinity for FlPK for its substrates, suggest
that it is involved in the metabolism of fructose. Similar

reasoning might suggest that the 6-FK is also involved in

 

the metabolism of free fructose, which it is not, as
indicated by two different approaches using mutants (Sec-
tion LL). However as found by the analysis of FlPK nega-
tive mutants, FlPK is instrumental in the metabolism of
free fructose (Section Li). As indicated by its lack of
induction by sucrose it is not involved in sucrose
metabolism under normal conditions (Section )4). The 6—FK,
as suggested by its higher induction by sucrose than by
fructose (and by the relative lack of induction of FlPK
by Sucrose) is involved in the metabolism of sucrose and
Possibly other di-, tri- and polysaccharides containing
fructose (Section h). The lack of coordinate induction

0f 6-FK and sucrase suggests that they are under the
control of different operons. However prior to the isola—

tion and analysis of mutants it was necessary to assume

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that free fructose was metabolized by two pathways, one
via FIP and the other via F6P. Subsequent to the estab-
lishment of the presence of FlPK it was shown that the
acyl phosphate, hexose phosphate:hexose phosphotrans-
ferase of Kamel and Anderson, which had instigated the
interest in FlP, had such a low affinity for fructose
that it could no longer be considered as an in 3119
source of FlP (6). Thus it was necessary to find an in
vim source of FlP in order to complete the FlPK pathway.
Completing the pathway would of course strengthen the
case for the involvement of FlPK in metabolism, which

in view of the existence of a complete and apparently

functional pathway via F6P, was somewhat doubtful.

1.3.3. OTHER INVESTIGATIONS or FlPK. Simultaneously
with the present investigation, Reeves gt a; (21) found

an FlPK in the anerobe, Bacteroides symbiosus, an organ-

 

ism which does not appear to have an F6PK. The FlPK has
a very high specific activity and is semi-constitutive.
The source of FlP for the enzyme is not known at present.
One idea (D. S. Hsu, personal communication) is that the
enzyme in ]_3_. s iosus is involved in the metabolism of
fructose by the parasitic amebas which feed upon this
OI‘Qanism, i.e., MP is produced in the cytoplasm of

the ameba and the bacteria (or their enzymes) further
metabolize it. As described in Section 14.2.14. FlPK has

8130 recently been found in E. coli, in which it appears

.1

 

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to function in an FlP pathway identical in many respects
to that in A. aerogenes (22).

The symmetry between the FlPK and F6PK reactions
suggested that metabolism of FlP via FlPK is essentially
a bypass of the F6PK reaction and that since F6PK has a
control function in glycolysis in most organisms (23), FlPK
might also exhibit similar properties for the Control of
fructose metabolism. The kinetics of the FIPK from g,

symbiosus have been examined and were found not to suggest

 

control properties (2h). H0wever the organism has no F6PK
for comparison. Recently,‘v. Sapico of this laboratory
has compared the kinetics of F6PK and FIPK from A,
aerogenes (11, 12). It was found that F6PK has the control

 

properties involving nucleotide phosphateS‘which are
common for this enzyme in other organisms but that FlPK
does not. The effectors of FlPK which were found acted
as competitive inhibitors of FlP (non-sigmoidal kinetics)
and included FDP, F6P and citrate (K1 values were h-S,
1.0 and 0.85 mM respectively). It is not at present
possible to propose a coherent control system in which
these effectors might be involved. It was apparent how-
ever that FlPK is not involved in a system modulated by
nucleotide phosphates. It was concluded (ll, 12) that
the difference in kinetics is a reflection of the fact
that, due to the FDPase reaction, F6PK would function

in a "cyclic ATPase" system if there were not special

 

 

 

 

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control mechanisms for inhibiting F6PK and presumably
FDPase. Since no pathway'which produces FlP from FDP in
A, aggggengs is known, and there is no reason to suspect
that there is such a pathway, FlPK would not be involved
in a cyclic.ATPase system, so that an analogy in kinetic
properties would not be expected. Thus the rationale
that the FIPK reaction is a bypass of a control reaction
in glycolysis and should require a similar control mecha-
nism is not substantiated. This suggests either that
possibly F6PK is not a control point in glycolysis under
certain conditions in this organism, and/or that fructose
metabolism has a special control mechanism, perhaps
involving enzymes other than FIPK. In the control of the
cyclic ATPase by F6PK and FDPase the primary function of
the control system for F6PK'would be to inhibit this
enzyme during gluconeogenesis. However in order to
achieve this control it is not necessary that F6PK be

a rate-limiting reaction in metabolism.viaguycolysis

as it is proposed to be in some other organisms (23).

It may be that F6PK limits the rate of glycolysis only
during gluconeogenesis and that the rate-limiting step
in the metabolism of glycolytic substrates occurs at
other points, for instance, the phosphoenolpyruvate-
dependent transport systems which are presently being

examined for possible control functions.

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1.3.h. SUPPLEMENTARY DISCUSSION —— REVIEW OF THE INVES -
TIGATION'OF MAMMALIAN FRUCTOSE METABOLISM. Investigation
of the metabolism of fructose via FlP has been primarily
confined to mammalian tissue in the past. any of these
investigations were instigated by the possible signifi—
cance of fructose in a treatment for diabetes or by the
possibility of explaining various types of fructosemia.
Illthough'we are not particularly familiar with the medi-
cal significance of this work, it produced an important
contribution to the understanding of carbohydrate metab-
olism. The major site of fructose metabolism in mammals
is the liver, which utilizes 30—50% of intravenously in-
Jected fructose and which can metabolize fructose 20 to
.ND'times faster than glucose (25, 26). The work of Cori
and ISlein (27, 28) suggested, and that of Leuthardt and
Tests (25, 29) and others (30) established, that a keto-
heXCflsinase forming FlP was present in liver. Cori gt al
(31) initiated an interesting series of experiments by
Pro‘fiding convincing evidence that F1? was further metab-
01ized via a magnesium-requiring phosphofructomutase,
whichconverted it to FéP, resulting in an accumulation
0f lrexose phosphates in partially purified liver extracts
in trm:presence of FlP and magnesium ion. Hers and Kusaka

(32) and Leuthardt and Taste (33) showed that though the

observations of Cori et al were correct, the reaction

actually involved cleavage, the in vitro formation of

 

 

   
 
 

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hexose phosphates from FlP being due to the enzymes of an
FlP aldolase pathway:
(1) l-Fructokinase

Fructose + ATP -——-> PIP + ADP
(2) Liver aldolase (aldolase B)

FlPis==$ Dihydroxyacetone phosphate + glyceraldehyde
(3) Glyceraldehyde kinase (triokinase)

Glyceraldehyde + ATP ———e-Glyceraldehyde—3-phosphate

+ ADP
(h) Liver aldolase (aldolase B)

Glyceraldehyde-B-phosphate + Dihydroxyacetone phos-

phate ;==E EDP
(5) Fructose diphosphate phosphatase (FDPase)

FDP -——> F6P + P1
(6) Phosphoglucose isomerase

F6? : 66?
This sequence of reactions is now a well established path-
way in liver metabolism (see references 3h and 35 for re-
views). .As established by Penarsky and Lardy (36),
Rutter g; g} (35) and others,the aldolase in liver which
'was responsible for the cleavage was a new enzyme (aldolase
B) which was active‘with FIP as well as FDP, and which was
distinct from muscle aldolase (aldolase A) which cleaves
FlP at a very low rate (see reference 37 for review). The
‘magnesium was necessary for a hexose diphosphate phospha-

tase activity in unpurified fractions which was active at

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pH 7 (38). A specific hexose diphosphate phosphatase
with a very alkaline pH optimum had been found previously
by Gomori (39). The transition in pH optimum which was
probably responsible for, or related to, the two forms

was found by Pontremoli e_t a_1 (39) and it now appears that
the PIP pathway in liver was the first example in which
the role of FDPase in metabolism was evident. In fructose
metabolism in A. aerogenes the role of FDPase also became
apparent (Section ll.2.ll). The GéP produced from fructose

may be converted to glycogen or released as glucose for

 

utilization in other tissues(30, 11.0). Three other path- ‘
ways for the utilization of glyceraldehyde have been (
SuSgested (l9) and pyruvate may also be a final product
(25) (see references 35 and Lil for reviews on liver
metabolism).
The metabolism of fructose in muscle is not well
understood. Muscle is not considered to be an important
Site of fructose metabolism (25). As indicated by labeling
experiments with whole animals, the liver converts most
of intravenously injected fructose into glucose which is
then utilized in muscle and other tissues (30). The keto-
heXokinase found in the liver was actually suggested by
the finding by Cori and Slein of a similar enzyme which
Produced HP in muscle (15, 27, 28). As shown by Hers
(30), this enzyme, unlike that in liver has a very high
Km for fructose of 0.19 M. There are also two types of

non-specific hexokinases in muscle which produce F6P (142).

¥ 4

 

 

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Silein, Cori and Cori (15) obtained from muscle an ammonium
srrlfate fraction which in the presence of ATP catalyzed a
disappearance of FlP and an appearance of FDP — an appar-
ent. FIPK activity. However their examination did not ex-
cltuie the possibility that the activity was due to the re-
actdions catalyzed by an PIP aldolase and glyceraldehyde
kina se or to other reaction sequences. In a reexamination
Here: (30) found that in crude extracts of muscle there was
a conversion of PIP to triose phosphate in the absence of
ATP’ and this conversion was not stimulated by.ATP -— an
apparent FIP aldolase. However there was in the extracts
no gglyceraldehyde kinase which was thought to be necessary
for the PIP aldolase pathway and the labeling pattern of
frucrtose-l-Clu incorporation into muscle glycogen indicated
that; cleavage does not occur (30). There has been consid-
eralale doubt in the past as to whether glyceraldehyde
lfiruase (triokinase) is absolutely necessary for the PIP
aldlease pathway (19) but subsequent results (see below)
huiicate that this is the case. Hers (30) also found that
incorporation of fructose into glycogen in muscle was al-
most completely abolished in the presence of a high con-
centration of glucose, an indication of fructose phospho-
rVlation by the hexokinases for which the phosphorylation
of fructose is competitively inhibited by glucose. Thus
the Present status of fructose metabolism in muscle is

that a slow rate of utilization occurs via a hexokinase

r..-

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'which produces P6P (30, h3). However even this is in
doubt (3) and may perhaps apply only to that component
of metabolism which is directed toward glycogen produc-
tion. The opinions of reviewers with respect to PlPK

in muscle and muscle metabolism of fructose in general,
have varied considerably in the past (3, 30, no, uh, AS).
There is substantial evidence suggesting that the l-
fructokinase activity in muscle is actually catalyzed

as a side reaction by P6PK and is not of significance

in vivo (3).

 

More recently, extensions of the previous work on
the PIP pathway in liver have been made‘which are of
interest‘with reapect to the metabolism of fructose in
muscle (see references 3h and 37 for brief reviews and
recent references). It has been established that the
three enzymes present in liver, l-fructokinase, trio-
kinase and aldolase B, occur together as a "cluster"
in various tissues. These tissues include the liver,
the small intestine and the renal cortex (3h). In
some forms of hereditary fructosemia, which are believed
to be due to a lack of aldolase B, the abnormalities of
the disease are apparently initiated by an accumulation
of P1P specifically in those tissues which normally
contain all of the three enzymes of the cluster (3h, 35).
The establishment of the three enzymes as a cluster

indicates that the PIP aldolase pathway occurs as

 

 

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a‘well defined metabolic unit in mammals so that it now
appears less likely that in a particular tissue, substi-
tution of one or more of the three enzymes would occur
to produce an altered version of this pathway. Thus
since it is now well established that muscle does not
contain aldolase B (35, ho), the finding by Hers men-
tioned above, of an apparent P1P cleavage at a rather
high rate in muscle was probably an artifact, due per-
haps to the presence of ATP in the crude extract which
was used. It therefore appears more likely that PIPK
does exist in muscle as proposed by Cori g; gl (IS).
The problem at present is to confirm that PIPK is pre-
sent in muscle and to find a suitable source of P1P in
muscle in order to complete the pathway. .As mentioned
above all investigations of which we are aware indicate
a lack of fructose metabolism in muscle. An important
point to note however is that only adult animals were
examined in these investigations. There is highly sug-
gestive evidence that fructose metabolism in the fetus
may be quite different from that in the adult. Investi-
gations by Hers and by Leuthardt (see reference 25 for a
review) of several species of mammals (including prelim-
inary'work on man) indicate that in the placenta maternal
glucose is converted to sorbitol by an aldose reductase.
The sorbitol is transported by fetal circulation to the

liver (presumably)'where it is converted to fructose.

.(W A

I \

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50
The fructose formed in the liver is apparently released
into the blood producing a higher concentration of fruc-
tose in fetal blood than in that of the adult. [In the
fetal blood of sheep 30-50% of the total reducing sugar
is fructose (lgz);] This is in contrast to the adult
animal in which intravenously injected sorbitol is con-
verted in the liver, first to fructose, then to glucose
which is released by the liver into the circulation (hO).
These results suggest that fructose may be metabolized
in a wider variety of tissues in the fetus than in the
adult. Furthermore it suggests that in these fetal
tissues metabolism occurs via a pathway which is directed
primarily toward production of energy directly from fruc-
tose, rather than toward conversion of fructose to glu-
cose, which appears to be the major function of the F1?
pathway in adult liver, aldolase B being particularly
adapted for condensation (37). This fetal pathway may
involve FlPK. If the existence of FIPK in muscle were
confirmed it would not be surprising to find that the
enzyme producing FlP in muscle is present in the fetus
but is induced only under particular metabolic situations
in the young and in the adult. Also supporting the
existence of a special fetal pathway for fructose metab-
olism is the finding (35, 37) that during the early stages
of development the fetal liver contains only a fetal

aldolase which is similar (or identical) to aldolase A.

f

l JI‘O‘ g "m 3‘: {W _. "-—

.
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we:

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Ez'aIicIase E

mid explain

tissues other

£215: of fruc

 

51

This aldolase is replaced almost completely in the liver
by aldolase B in the later stages of development. This
‘would explain why metabolism of sorbitol by the fetal
liver would result in the production of fructose rather
than glucose. It also suggests the existence in fetal
tissues other than the liver, of a pathway for the metab-

olism of fructose which does not require aldolase B.

5
'_.':..es C

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52
1.14. METHODS

1.h.1. GROWTH OF CELLS.AND PREPARATION OF CRUDE EXTRACTS.
Cultures of Aerobacter aerogenes, PRL-R3, were routinely
grown to the early stationary phase in 12 to 18 hours on
0.5% carbohydrate (autoclaved separately) in 500 ml of
mineral medium in 2800 ml cotton-plugged Fernbach flasks
on a rotorary shaker (about 150 RPM) at 32°C. The mineral
medium contained: 0.71% 'Naznpou, 0.15% KHZPOLL, 0.3% (NHLL)2

SOLL’ 0.009% MgSOu and 0.0005% FeSOu'7H20. Cells were har-
vested by centrifugation at 0°C (all subsequent steps

‘were at 0-5°C), washed once in distilled water (about 250
ml per gram of cell paste), suspended in water (3.0 ml

per 1.0 gram of cell paste). Cell suspensions were soni-
cated 5 to 20 minutes in a Ratheon 10 Kc sonic oscillator
until approximately 95% breakage had occurred as judged by
phase contrast microscopy. For exploratory assays, cells
and crude extracts‘were used within 6 hours of harvest.

For purification, cells and crude extractS'were stored at

-2o°c.

l.h.2. ASSAYS.

l.h.2.A. GENERAL TECHNIQUES FOR ENZYMATIC ASSAYS. All
continuous spectrophotometric assays used throughout this
work, unless noted otherwise, were made in a total volume
of 0.15 ml, in 0.5 ml cuvettes (1 cm light path), at pH
7.5, at 25°C, on a Gilford recording spectrophotometer, at

-
. 3' cu:

 

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11.1 ..., w-

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30“! 1" C

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53

3h0 nm if TPNH or DPNH absorption was determined. The buffer
used was glycylglycine, pH 7.5,‘with 10 pmoles per assay for
data in Sections 1 and 2 and 20 pmoles per assay for data in
Sections 3 and h. All components which have appreciable
buffering capacity‘were neutralized to pH 7.5 with NaOH or
HCl. An excess of coupling enzymes was used. The amount

of each coupling enzyme is not given since the values vary
depending on the preparation used and must be established
for each new set of assays. Uhless noted otherwise,rates
were taken in the linear region of the assay, usually
between the third and 15th minutes. Listed with each

assay below is the blank which gives the highest rate in
crude extracts and which was used for routine assays. The
other possibly important blankS‘were checked for appreciable

activity using crude extracts.

l.h.2.B. ENZYME UNITS. Throughout this work, unless stated
otherwise, all rates for continuous spectrophotometric
enzymatic assays, in which pyridine nucleotides are involved,
are given in terms of pmoles of pyridine nucleotide produced.
Specific activities (S.A.) are given as pmoles of product
formed per minute per milligram of protein unless noted‘
otherwise. For assays of crude extracts it can generally

be assumed for an assay coupled with glucose-6-phosphate
dehydrogenase gives two moles of TPNH per mole of 66? pro-
duced. The routinely used assay for FlPK (and the similar

assay for aldolase) coupled with aldolase,«pglycerophosphate

I. C.

‘11 K! I". 'A‘

 

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5L1

dehydrogenase and triose phosphate isomerase also gives 2
moles of DPN per mole of FIP (or FDP). The conversion
factor used for pyridine nucleotide-dependent assays was

0.11.1 AO.D. per 0.01 ,umole per 0.15 ml assay. For other

3110

types of assays conversion factors are given with the

assay description.

1.Li.2.C. ENZYMATIC ASSAYS. Numbers are pmoles per 0.15 ml
and components are listed in the order of addition to the
cuvette following addition of water and glycyl glycine.

1. FlPK (aldolase-d-glycerophosphate dehydrogenase) - This

 

assay was used for FIPK unless stated otherwise. DPNH
0.007, a-glycerophosphate dehydrogenase plus triose phos-
phate isomerase, aldolase, MgCl2 2.0, ATP 1.0; extract,
PIP 1.0: blank-minus ATP.

2. FlPK (pyruvate kinase-lactic dehydrogenase) - DPNH 0.007,

 

lactic dehydrogenase plus pyruvate kinase, MgClg 2.0, ATP 1.0,
PEP 0.5, extract, FIP 1.0; blank—minus HP. The lactic
dehydrogenase used in this and similar assays contains the
pyruVate kinase required for the assay as a contaminant.
However it also contains contaminating aldolase, tx—glycero—
Phosphate dehydrogenase and possibly other enzymes. It
contains a very low level of an enzyme which apparently
catalyzes the DPNH dependent reduction of FlP, which is of
interest since this preparation is derived from rabbit

muscle. These impurities make it difficult to interpret

10‘" activities obtained in the absence of various components

. JV

'4!

~ ”-flu-m-‘l ITIW

,I’w

a... 01.12 I.) 7"

uh u

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"1.; 2341

“‘

extracts a"

is.

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h.

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55

for the FlPK assay (such as those in Table 1.11).

3. HP or FDP Aldolase ( «X—glycerophosphate dehydrogenase) -
DPNH 0.007 a—glycerophosphate dehydrogenase plus triose phos-
phate isomerase, extract, FlP or FDP 1.0; blank—minus FlP

or FDP; addition of MgClg, 0.5, had no effect using crude
extracts and HP or FDP.

11. PIP or FDP Aldolase (hydrazine) - hydrazine sulphate

 

0.2, extract, HP or FDP 1.0; blank-minus extract; O.D.
taken at 2110 run; units are AO.D.2L'_O/min; addition of
MgCl2 0.5 had no effect using crude extracts and PIP or
FDP.

5. FéPK (aldolase-d-glycerophosphate dehydrogenase) — same

 

as FIPK (aldolase-a—glycerophosphate dehydrogenase), minus
FlP plus F6P 1.0; blank-minus ATP.

6. Glyceraldehyde kinase (pyruvate kinase-lactic dehydro-
genase) - same as FlPK (pyruvate kinase-lactic dehydro-
genase), minus FlP plus DL-glyceraldehyde 2.0; blank-minus
ATP.

7. 6-Fructokinase (phosphoglucose isomerase - glucose-6—

PhOSphate dehydrogenase) - This assay was used for 6-FK

 

unless stated otherwise. TPN 0.01, glucose-é-phosphate
dehYdrogenase, phosphoglucose isomerase, IVIgCl2 2.0, ATP 1.0
extract, fructose 1.0; blank—minus ATP.

8- Mockinase (glucose-6-phosphate dehydrogenase) - TPN
0'1, glucose-é-phosphate dehydrogenase, Mng2 2.0, ATP 1.0

€Xtract, glucose 1.0; blank-minus ATP.

 

 

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56
9. Sucrase (glucose oxidase) - indicator dye (0.01 ml of a
solution containing one vial of the glucostat dye reagent
per 5 ml water), glucose oxidase (0.01 ml of a solution con—
taining one vial of the glucostat enzyme per 2 ml water),
extract, sucrose 1.0; blank-minus sucrose. The dye concen-
tration is critical due to precipitation; extract concentra-
tion is critical due to reducing material in crude extracts;
extracts containing mercaptoethanol cannot be used. 0.D.
taken at A30 nm, o.uu AsO.D.h30/O.01 pmole/O.15 ml.
10. Fructose-1,6-diphosphate end-point assay (aldolase-
glycerophosphate dehydrogenase) - DPNH 0.007, sample, «—
glycerophosphate dehydrogenase plus triose phosphate iso-
merase, initiated with aldolase; blank-minus sample; 2

moles DPN/mole FDP.

1.h.2.D. OTHER ASSAYS. Fisk-SubbaRow assay for free phos-
phate (h8); acid hydrolysis for total phosphate (uq); Roe
test for fructose (50); anthrone assay for carbohydrate
(51). Unless specified otherwise protein was determined
‘with the 0.D. 260-280 nm method (52) using a nomogram

supplied by Calbiochem.

l.h.3. ENZYME PURIFICATION.

l.hs3.A. GENERAL ENZYME PURIFICATION TECHNIQUES. For all
purifications in this work involving ammonium sulfate
fractionation the technique was as follows. The crude

extract was diluted with water to give 10 mg protein per

, ...-......”

 

 

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:. as .t. :2.
I u . ‘f
.1211: su..
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a“?

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57

ml as determined by the 0.D. 260-280 nm method. Crystalline
ammonium sulfate was added to give a concentration of 0.2
M. A 20% volume of 2% protamine sulfate, pH 7.0, was
added and the precipitate was removed by centrifugation.
Ground crystalline ammonium sulfate was added slowly with
stirring to precipitate protein which was removed by
centrifugation and dissolved in water to give 20 to ho mg
protein/ml. The amount of ammonium sulfate is expressed
as "percent saturation". Total grams of ammonium sulfate
(including the initial 0.2 M ammonium sulfate) per m1 of
preparation for the percentages used were: 0% (0.2 M) -
0.026 g, 30% - 0.158 g. 35% - 0.186 g, 10% - 0.218 g. 55%
-0.318 g, 60% - 0.351 g, 80% - 505 g, 100% - ammonium

sulfate was added until there was an excess in the breaker.

l.h.3.B. PURIFICATION OF FlPK. For the routine procedure
a crude extract of fructose-grown CCIIS‘WBS treated with
protamine sulfate. From the supernatant a 35-55% ammonium
sulfate fraction was made and heat treated in a test tube
or small flask for 2 to 5 minutes in a 52°C water bath
with gentle shaking. The heat treated material was centri-‘
fuged and the supernatant was chromatographed on Sephadex
G100 in 0.02 M sodium phosphate, pH 7.5, using a sample
volume of about 10 percent of the bed volume of the column.
The active fractions were pooled and frozen. To a 2.0 ml
sample of the pooled fractions 0.2 m1 of calcium phosphate

gel (62 mg/ml of suspension) was added, stirred, and

r'\ 0' W‘

 

ireiiately
tapers-tare
elitei with
s:s;er.si3n I
final prepar
FIPassajrs i

...ctia: was

1 r .1
292.?13 fie“
‘. ‘2 l, r: "
l' ‘U‘. ./

‘.

I: ‘_‘ a: .

.. 1 tie
a“ ‘ A a
9.363‘ ‘1
I‘l“.n‘

fierce” 1
3: 2 3.1 0“ v

58

immediately removed by centrifugation for 2 minutes at room
temperature in a clinical centrifuge. The precipitate was
eluted with 1.0 ml of 0.1 M sodium phosphate, pH 7.5, the
suspension was centrifuged and the supernatant taken as the
final preparation. If necessary for end-point assays (for
FlP assays in the presence of glucose),the Sephadex G100
fraction was purified with a pH treatment. The Sephadex
fraction was adjusted to pH 5.8 (pH meter standardized at
25°C, sample at 0°C) with dilute acetic acid, immediately
centrifuged, and the supernatant was immediately adjusted
to pH 7.5'with 0.1 M NaOH. The pH-treated sample was then
treated with calcium phosphate gel as described above.

For the most successful DEAE cellulose purification
of FlPK the sample consisted of one m1 of a five-fold con-
centrated fraction from Sephadex G100 chromatography.
Lypholization was used for concentrating and resulted in a
90 percent recovery of activity. The sample was adsorbed
on 8 ml of DEAE cellulose in a 0.7 cm x 20 cm column which
was equilibrated with 0.05 M sodium phosphate, pH 6.32, plus
0.01 MngClg. The column was eluted with a 60 ml linear gra-
dient of from 0 to 0.6 M NaCl in the same buffer as used for
equilibration. FlPK was eluted (60 percent recovery of
activity) in the 21 to 25 ml volume range, one m1 ahead
of a major protein peak. The purification for this step
‘was 2-fold; the final purification was hO-fold. Other
variations of ion-exchange chromatography which were found

unsatisfactory in terms of either recovery of activity or

‘T

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59
purification included the following. (1) DEAE cellulose
eluted with gradients of NaCl in, (i) 0.05 M sodium phos-
phate (pH 6.5, 7.5 or 8.0), (ii) 0.05 M sodium phosphate
(pH 7.5) plus 0.05% mercaptoethanol, and (iii) 0.05 M
sodium phosphate (pH 7.5) plus 0.01 M'MgCla. (2) Carboxy—
methyl cellulose eluted with a gradient of NaCl in 0.05
M sodium phosphate (pH 6.5 or 7.5).

l.h.3.C. PURIFICATION OF 6-FRUCTOKINASE. For the single
experiment run a crude extract was treated with protamine
sulfate and 0-35%, 35-55% and 55-100% ammonium sulfate
fractions'were made. The 0-35% fraction,containing 85%

of the original activity,was chromatographed on Sephadex
0100 in 0.02 M sodium phosphate, pH 7.5, to give a single
peak in the region where glucokinase (M.W. 70,000) appears.
Total recovery of activity after chromatography was about
85 percent of the activity in the crude extract. A frac-
tion from the center of the peak (35-fold purified) was
tested as described in Table 1.9. The phosphoglucose
isomerase-glucose-6-phosphate dehydrogenase-coupled assay
was used during purification. Specific activities of the
fractions in pmoles TPNH/min/mg protein were: crude
extract, 0.0093; 0-35% ammonium sulfate, 0.019; peak frac-
tion of the Sephadex material, 0.33.

. :1

(FT—

: )1 1
In" unfit my. as» 'm—‘1

 

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til-.2 la. a;
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60

l.h.h. PREPARATION.AND IDENTIFICATION OF THE PRODUCT OF
FlPK. The reaction mixture contained in a total volume of
5 ml: 50 umoles ATP, 100 umoles MgC12J 57 umoles FlP, 0.90
pmole DPN/min of FlPK and no buffer. The FlPK used was the
hO-fold purified preparation obtained by chromatography on
DEAE cellulose as described in Section l.u.3.A. The acid
produced by the reaction mixture was titrated at pH 7.5
.with a Sargent pH Stat using 0.027 M NaOH. The reaction
mixture was incubated at 25°C for 90 minutes when acid pro-
duction ceased. The pH of the mixture was adjusted to 8.h
with dilute ammonium hydroxide, the volume was adjusted to
15 ml and the sample was chromatographed on a 10 x 0.7 cm
column of Dowex 1 resin (100-200 mesh) equilibrated with
0.001 M'NHEOH. A gradient elution was made with a Technicon
'Autograd gradient producer containing the following (150 ml
of solution per compartment): compartment no. I - 0.001 M
NHEOH: no. 2 - 0.025 M NHuC1,~0.001 M NHEOH, 0.01 M
K?BbO7: no. 3 - 0.025 M NHhCI, 0.0025 NHhOH; 0.00001 M
KthO7; nos. h, 5 and 6 - 0.01 M HCl, nos. 7 and 8 - 0.02
M HCI, 0.02 M KCl. Following the gradient elution the
column was eluted with 0.02 M HCl, 0.2 M KCl in order to
lelute ATP. The method is a variation of that of Khym and
Cohn (53, 5h, 55). Ten ml fractions were taken. Carbohy-
drates were determined using the anthrone assay, and
nucleotides were determined by adsorption at 260 nm.

All of the fractions containing FDP were pooled (250
m1 total) and a 100 ml sample was lyophilized to dryness.

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The lyophilized powder was added to an acid phosphatase
reaction mixture (3.h ml total volume) containing 50 pmoles.
MgClz, 250 pmoles sodium acetate, pH h.6, and 10 mg wheat
germ acid phosphatase. The reaction was incubated at 25°C
for 12.5 hours, at which time total phosphate was equal

to free phosphate, and the reaction mixture was deionized
by passage through a 30 x 0.7 cm column of ion retardation .
resin (BioRad AG-11A8) eluted with water. The sample was

compared with D-fructose, L-sorbose, D-tagatose and D-

 

psicose by paper chromatography using Watman no. 1 paper
eluted with water saturated phenol and developed with
silver nitrate (56)- For the comparison using the cystein -
sulfuric acid procedure (57) the reaction mixture contained
0.5 ml water plus sample, 0.1 ml 25% cystein-HCI and 6 ml
25 M HZSOh‘ The mixture was incubated at 25°C for 2h hours
and the absorption spectra from 300 nm to 700 nm was taken
‘with a Cary Model 15 spectrophotometer. The spectrum of
the sample was identical to that for fructose, similar to
that for tagatose and psicose and different from that for
L-sorbose. The time for half-maximal development of 0.0.
at hl2 nm was determined by initiating the cystein-
sulfuric acid reaction with sugar in a cuvette at 25°C in

a Gilford recording spectrophotometer and allowing the
reaction to proceed to completion (maximum.O.D.h12) after
which there was a very slow drop in absorbance. Using

different concentrations of sugars in this determination

 

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62

it was found that the time for half-maximal development
of O'D'h12 was a constant, independent of the sugar concen-

tration.

l.h.5. REAGENTS. D-psicose was prepared from D-psicofuranine,
a gift of the Upjohn Company, by acid hydrolysis in 0.5 M
H2soh for 17 hours at 25°C (58). The sugar was freed of
nucleotide and salts by chromatography of the neutralized
reaction mixture through a 10 cm x 0.7 cm column containing
Dowex 50W-x8 and Amberlite CG-hB (in proportions as as to
give a one to one cation to anion exchange capacity)

eluted with water. D-tagatose was a gift of Dr. H. Lardy.
F6P, FlP and FDP were obtained as barium salts from either
Boeringer or Calbiochem and were converted to the sodium
salt by treatment of a 0.1 M solution with three-fourths

of its volume of Dowex Sow-x8. Glucose-6-phosphate
dehydrogenase (yeast),‘i-glycerophosphate dehydrogenase

plus triose phosphate isomerase (rabbit muscle), aldolase
(rabbit muscle) and phosphoglucose isomerase (yeast) were
obtained from Sigma, Calbiochem and Boeringer in the highest
purity available. Lactic dehydrogenase plus pyruvate kinase
(rabbit muscle), acid phosphatase (wheat germ) and the
Glucostat reagent were from Worthington. 2-keto-3-deoxy-
gluconate (sodium salt) were the gift of M. Roseman.

Calcium salts of 2~ketogluconate and 5-ketog1uconate were
from N.B.C. and were converted to the sodium salts before

use. Fructose and other sugars were obtained from Pfanstiehl,

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63

protamine sulfate from Calbiochem or Sigma, ATP, TPN and
DPNH (sodium salts) from Pabst, phosphoenolpyruvate,
tetracyclohexylammonium salt, from Calbiochem, glycylglycine
from N.B.C., potassium gluconate from Pfizer, and DEAE
cellulose from Sigma. DEAE cellulose was washed with HCl
and NaOH (59). Calcium phosphate gel (60) was prepared by
0. Allison.

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SECTION 2. ISOLATION AND CHARACTERIZATION OF
A HEXOSE PHOSPHATE.:HEXOSE (H20)
PHOSPHOTRANSFERASE OF UNKNOWN FUNCTION

SUMMARY: A hexose phosphate:hexose (H20) phosphotransferase
'which catalyzes the formation of F1? was'identified and puri-
fied from extracts of A, aerogenes.- Establishment of the re-
actions catalyzed andpreliminary kinetic data indicated that
the enzyme has a relatively high affinity for glucose (Km =
0.002 M) and that besides hexose phosphotransferase reactions
it catalyzes, at relatively high rates, phosphorylation with
phosphoramidate and the hydrolysis of the monophosphates of
fructose and glucose. Temperature-dependent modifications
of chromatographic and kinetic properties were observed but
these did not result in a change in hydrolytic yg_hexose
phosphotransferase activities. Although it was initially
thought that this enzyme might have a role in fructose metab-
olism such a role was not substantiated in further investiga-
tions.
2.1. INTRODUCTION

As presented in Section 1, there is evidence that FlP
is an intermediate in fructose metabolism. In this section
we describe the isolation and characterization of a hexose
phosphatezhexose(H20) phosphotransferase (hereafter called
the "H-phosphotransferase") which catalyzes the formation

of FlP. It was initially proposed that this enzyme might
be the ig_vivo source of FlP in.the metabolism of fructose

(8). It exhibits several unique properties which are of

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65

interest with respect to some of the other phosphotrans-
ferases which have been found recently in.A, aerogenes and
other bacteria. However it was ultimately established by a
variety of criteria that the H-phosphotransferase is not in-
volved, at least specifically, in the metabolism of fruc-
tose. Although the properties of the enzyme suggest that

it has some role in hexose metabolism, its exact function

in_vivo, like that of similar enzymes, is unknown.
2.2. RESULTS

Note: The different reactions catalyzed by the H-phospho-
transferase and the assays used are referred to in the for-
mat¢—— "phosphoryl donor, phosphoryl acceptor:product deter-
mined". For instance, for glucose production from the
hydrolysis of GlP the format is G1P,H20 : G; for production
of GlP and G6P (simultaneously) from glucose and FlP it is
FIP,G : GP; for FlP production from GlP and fructose it is

GlP,F : FlP. See Methods for other assays.

2.2.1. DETECTION OF GLUCOSE-l-PHOSPHATE DEPENDENT FORMA-
TION OF FRUCTOSE-l-PHOSPHATE. A survey for FlP produc—
tion catalyzed by crude extracts of fructose-grown cells
was made using an assay containing FlPK, aldolase and
cx-glycerophosphate dehydrogenase. Of the phosphate
donors tried only glucose-l-phosphate and, to a lesser

extent, glucose-6-phosphate produced detectable activity.

 

   
  

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66

Other donors tried included acetyl phosphate (0.0066 M),
phosphoenolpyruvate (0.0033 M), carbamyl phosphate (0.0066
M), mannose-6-phosphate (0.0066 M), ribose—5-phosphate
(0.0066 M), phosphocreatine (0.0066 M), 3-phosphoglycerate
(0.0066 M), inosine triphosphate (0.0066 M) and IJLaae
glyceraldehyde-3-phosphate (0.0033 M). .ATP, which was
required for the FlPK in the assay, gave a very low level

of activity which was probably due to the 6-fructokinase

 

activities present (Section I). The apparent GlP,F : FlP
activity was constitutive and had a reasonably high specif-
ic activity (Table 2.1). Using crude extracts of fructose-
grown cells, a decrease in fructose concentration from
0.066 M to 0.0066 M gave a 2-3 fold reduction in activity.
Using crude extracts the assay for FlP production was non-
linear, both with respect to time, there being a lag period
of about 10 minutes, and with respect to extract concentra-
tion. The activity in the absence of fructose, the rate

of which was 50-80% of that in the presence of fructose,
consisted of a component catalyzed by DPNH oxidase and

a component catalyzed in the presence of 61? without
fructose. The latter is presumably due to the conversion
of G1? to F6P or FDP via glycolytic enzymes. Due to

the presence of these and other enzymes in crude extracts,
it wms not possible to distinquish reliably between
formation of FlP and F6P. Using crude extracts the

assay was essentially a semiquantitative indicator, and

67

Table 2.1. APPARENT FlP PRODUCTION BY CRUDE EXTRACTS.

The GlP,F : FlP assay was used. The fructose concen-

tration was 0.066 M; the GlP concentration was 0.0066 M.
Growth Substrate Specific Activity

(pmoles DPN/
min/mg protein)

Fructose 0.067
Glucose 0.033
L-Sorbose 0.105
annose 0.073
Mannitol 0.068
Sorbitol 0.107

Nutrient Broth 0.087

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68
due to the nonlinearities of the assay the rates were
restricted to a relatively small range in order to ob-

tain comparable results for different crude extracts.

2.2.2. PURIFICATION OF THE H-PHOSPHOTRANSFERASE AND IDEN-
TIFICATION or REACTIONS CATALYZED. Using the 011-31“ : FIP
assay ammonium sulfate fractionation separated the three
activity components which contribute to the assay as fol-
lows. "DPNH oxidase was substantially inactivated by
protamine sulfate treatment and the remainder was found
predominantly in the 0 to 35% fraction. The activity
dependent on G1? and independent of fructose was found
predominantly in the 35-65% fraction and the activity
dependent on both fructose and GlP (Table 2.2) was found
primarily in the 65-100% fraction. As will be shown, the
H-phosphotransferase could also be assayed using the
reverse reaction in which GlP and G6? are simultaneously
produced from FIP and glucose (the FlP,G : GP activity).
In crude extracts the FlP,G : GP assay gave specific
activities of 0.002 to’0.01 umole TPNH/min/mg protein.
The FlP,G : GP activity‘was also isolated in the 65-100%
ammonium sulfate fraction (Table 2.2).' Chromatography of
ammonium sulfate fractions on Sephadex 0100 in 0.02 M
sodium phosphate, pH 7.5, resulted in single activity
peaks using the FlP,G : GP assay and in the one case tried,

the GlP,F : FlP assay.

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69
Table 2.2.

PHOTRANSFERASE.ACTIVITIES.

ANNONIUM SULFATE FRACTIONATION FOR H-PHOS-

Crude extracts were treated

with protamine sulfate prior to ammonium sulfate fractiona-

tion.

A. Using the GlP,F : FlP assay:

 

 

 

 

 

Total
Step Specific Activity Units Fold Recovery
(pmoles DPN/ (umoles/ (%)
min/mg protein) min)
Crude extract 0.102 11.3 1 100
Ammonium
sulfate:

0-35% 0.0082 0.12 0.08 1
35-65% 0.019 0.72 0.19 6
65-100% 0.1M 2.5 l.h 22
B. Using the FlP,G : GP assay:

Total
Step Specific Activity Units Fold Recovery
(pmoles DPNH/ (umoles/ (%)
min/mg protein) min)
Crude extract 0.005h 16.2 1 100
Ammonium
sulfate:

0-35% 0.000 0.0 0.00 0
35-55% 0.000 0.0 0.00 0
55-67% *0.000 0.0 0.00 0
67-100% 0.021 8.2 3.96 51

 

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During purification (see Table 2.3 for the complete
purification procedure), various activities were found
in H-phoSphotransferase fractions some of which were
apparently catalyzed by the same enzyme as indicated by
coincidence of elution positions in chromatography. In
the first series of experiments, the FlP,G : GP assay
was used to purify the activity in a crude extract of
fructose-grown cells to the ammonium sulfate stage. The
65-100% fraction was chromatographed on Sephadex 0100
(Figure 2.1) and the elution profile was assayed for the
’FlP,G : GP, GlP,G : G6P, and GlP,F : F6P activities, all
of which were found to coincide. The peak fractions of
this elution profile were then chrmmatographed on DEAE
cellulose using stepwise elution and the fractions were
assayed for the GlP,F : FlP and FlP,G : GP activities
‘which‘were found to coincide (Figure 2.2). This step
removed phosphoglucose isomerase which would interfere
in further characterization and it also substantially
reduced the level of phosphoglucomutase. All of the
assays made with the gel—filtered and DEAE cellulose-
purified fractions were linear with respect to time and
enzyme concentration, and with the exception of phospho-
glucomutase activity the blank values were low; The
peak fraction from the DEAE cellulose elution profile
(Figure 2.2) was used to establish the identity of some
of the reactions catalyzed by the H-phosphotransferase.

As shown below, in the presence of GIP and fructose

 

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72

Figure 2.1. COINCIDENCE OF H-PHOSPHOTRANSFERASE
ACTIVITIES —— GEL FILTRATION. A 10 m1 aliquot of a
67-100% ammonium sulfate fraction was chromatographed
on a 200 ml bed of Sephadex GIOO in 0.02 M sodium
phosphate buffer pH 7.5. Assays: (l) FlP,G : GP (0);
(2) GlP,G : G6P (U); (3) GlP,F : F6P (A); (u) phospho-
glucomutase (o). Phosphoglucomutase blanks are
subtracted where necessary. Assays were as in Methods
except that the GlP,F : F6P assay contained 0.012

M fructose (twice that used for data in Table 2.3).

73

Figure 2.1 .

 

 

 

 

 

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VOLUME ( ml)

 

 

 

 

Figure 2.2. COINCIDENCE OF H-PHOSPHOTRANSFERASE
ACTIVITIES —— DEAE CELLULOSE. A sample (1.0 ml)
from the peak fraction in the elution profile shown

in Figure 2.1 was adsorbed on a 1 m1 bed of DEAE

 

cellulose (2 cm x 0.5 cm) equilibrated with 0.02 M
sodium phosphate,pH 7.5,and eluted with 3 m1 of 0.05
M NaCl in buffer and 2 m1 of 0.2 M NaCl in buffer.
Assays: (1) FlP,G : GP (A); (2) GlP,F 2 FlP (O);
(3) phosphoglucose isomerase (D). The 2—2.8 m1
fraction was used for Characterization. The
Chromatography was perfdrmed at room temperature

(approximately 25°C).

75

Figure 2.2.

 

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76
the primary product was FlP. The assay for FlP formation
(Table 2.h) was relatively free of reactivity toward F6P
so that no more than 6 percent of the activity observed
with this assay could have been due to F6P formation.
In the FlP,G : GP reaction a mixture of GlP and G6P was
formed (Table 2.5). There was a low level of phospho-
glucomutase in the H-phosphotransferase preparation
so that in the absence of added phosphoglucomutase some
of the GIP was also detected. In the absence of magne-
sium, which was almost absolutely required for phospho-
glucomutase activity (both for that in the added pre-
paration and that in the phosphotransferase preparation),
the rate in the absence of phosphoglucomutase decreases
somewhat to a level which is probably the true rate of
G6P formation. The H-phosphotransferase itself did not
appear to require magnesium in this reaction as indicated
by product accumulation in the absence of magnesium,al-
though it cannot be ruled out that magnesium might have
some influence on the rates. Using assays similar to
those presented in Tables 2.h and 2.5, all of the phos-
photransferase reactions (for which it is possible to
provide enzymatic assays) involving the l- and 6-mono-
phosphates of glucose and fructose and the free sugars
were examined. In this experiment it was also found
that the H-phosphotransferase catalyzes a GIP phosphatase

reaction. The results of these assays are summarized in

 

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79

Table 2.6. At saturating fructose, formation of FlP was
the most rapidly catalyzed reaction. F6P was formed at
about one-third the rate of FlP. The rates given in
Table 2.6 may not necessarily be completely independent
of the techniques used to measure them. For an assay in
which two products are formed, both of which can also act
as substrates, but only one of which is removed by the
coupling reactions used in the assay, it is possible that
the accumulated product has some influence on the rates.
Complete linearity of the time course of the assays
suggested that this was not the case. However the lack
of discernible regularities in rates and product composi-
tions(Table 2.6) suggests that the results may be only
a preliminary reflection of a more complex kinetic system.
The affinities for substrates in several reactions are
given in Table 2.7. The high Km for fructose provides
the first factor militating against the involvement of
this enzyme in the 13 gigs production of FlP.

The second factor militating against such involve-
ment is the high level of sugar phosphate phosphohydrolase
activity which appears to be associated with the H-phos-

photransferase. The enzymatic assay for GlP,H20 : G
activity in Table 2.6 indicated a level of phosphatase

activity much higher than that of all the hexose phospho—

transferase reactions except the GlP,F : FlP reaction.

 

 

80

Table 2.6. PRODUCTS FORMED IN H-PHOSPHOTRANSFERASE
REACTIONS. Procedures were similar to those used in
Tables 2.h and 2.5 using the same enzyme preparation.
See Methods for assays used. Sugar phosphate concen-
trations were 0.0066 M (except F6P, 0.0019 M) and
sugars, 0.066 M. All of the possible (expected) H-
phosphotransferase reactions for the substrates

used are listed but only certain ones could be
assayed. Rates are given as pmoles of TPNH formed/
min/m1, corresponding to umoles of determined product
formed,except for FlP for which pmoles of DPN/a/min/ml

are given corresponding to pmoles 0f FlP/min/ml.

81

Table 2.6

 

 

 

 

Reactants Product Determined
(pmoles/min/ml)

Phosphoryl Phosphoryl . GIP 1 G6P FlP F6P Glucose

donor acceptor
i/

GIP Fructose .-— 1—— 0:337 0.092 -—
GlP Glucose -—- 00128 —— —— ——
G6P Fructose ——- ——» 0°055 -—- -—-
G6P Glucose -— —— —— —— _—
FlP Fructose 4— ——1 __. 0.019 ——
FlP Glucose 0.061 0.0u5g/ —_. -—- -—-
F6P Fructose ——» —— 0.016 -— -—
F6P Glucose 0.037 0.033g/ .—_ __ ...
01p H20 —_ .... — —— 0.11203/

 

 

 

 

 

 

 

2/

At saturating fructose the rate was 0.93 pmole DPN/2

min/ml.

G6P production is assumed to be the rate in the
absence of phosphoglucomutase and is slightly
higher than that in the absence of both phosphoglu-
See Table 2.5 for example.

comutase and MgClZ.

The specific glucokinase assay was used.

 

'3‘
1.”...

 

 

.11.. A
A-.
. p C
Ii-fiv
...1 c
.. .2 _

V“: n‘... ‘

..o ..

D. 9%: g .

an”:

..'

O-‘a

1.3
fl

Table 2.7.

SUBSTRATE AFFINITIES.

82

Procedures were as in

Table 2.6, except for the GlP,H20 : G (hexokinase) assays

which were made using a similar DEAE cellulose purified-

preparation.

 

 

 

 

 

 

 

 

 

Assay Substrate Km(M)
Phosphoryl Phosphoryl Product(s)

donor acceptor assayed

GIP Fructose FIP GIP 0.0005 "“

GIP Fructose FlP Fructose 0.07

GIP Fructose F6P Fructose 0.05

FlP Glucose GIP + G6P FlP 0.002 1‘"

FlP Glucose GlP + G6P Glucose 0.002
L_—‘EIP H20 G GIP 0.00022

 

 

:1;

this concentration.

Approximate value, assay gave half—maximal velocity at

, . 3"—
. ‘Léi‘bxxsw—J—‘J

  

" v
“Guav‘s‘
13:...”6.

“a“ '-
-.~"‘:

0.. ‘

 

83
The Km for GIP (Table 2.7) in the phosphatase reaction
was about the same as that in the GlP,F : FIP reaction.
Examination of phosphatase reactions (Table 2.8) indi-
cated a possible specificity for the l— and 6-monophos—
phates of glucose and fructose. Using a gel—filtered
preparation (Table 2.9) it was found that enzymatic
and non-enzymatic GIP phosphatase assays gave equiva-
lent results and that there was only a low level of
p—nitrophenylphosphate phosphatase activity in the
preparation. It was also found using Sephadex-purified
preparations that acetyl phosphate, phosphoenolpyruvate
and pyrophosphate did not serve as phosphoryl donors
when substituted for FlP in the FlP,G : GP assay at
the same concentration. Sodium fluoride, 0.02 M, and
ammonium sulfate, 0.05 M, inhibited completelyaboth the
GlP,H20 2 G (hexokinase) assay and the FlP,G : GP assay,
although the coupling enzymes were active under these
conditions. Sodium phosphate, 0.012 M, had no effect
on either of these two assays. In several different
attempts (Methods) to prepare phosphorylated products
by incubating partially purified H-phosphotransferase
Preparations with sugar phosphates and sugars, only
Sugars were found in the product mixtures. This was
Probably due to incubation for too long a period or
Possibly to lack of a unrecognized component which was

Present in the continuous assays,which did accumulate

nylon-I50

‘mut

 

o..-

“-

7: 0

 

8h

Table 2.8. SUBSTRATE SPECIFICITY IN PHOSPHATASE
REACTIONS. A DEAE cellulose-purified fraction (the
slow peak at 0°C) dialyzed against 0.00 M Tris-HCI
pH 7.5, was used. Substrate concentration was 0.001

M and free phosphate was determined.

Substrate Rate Rate
m1£7$31§36561n1 (% of GIP)
Glucose-l-phosphate 2.39 100
Glucose-6-phosphate IoSA 6A
Fructose-l-phosphate 1-39 58
Fructose-6-phosphate 1.30 55
Mannose-6-phosphate 0-33 13
Ribose-S-phosphate 0.18 7

F1"uctose-l,6-diphosphate 0-28 11

a: '9101—.

‘5’” ‘u ——-j..

0.

,._.__. -...

(-

AI\.;1 my,-

23-?

Q

f\_)

:a' ‘
P “:H

-«_...... .8]

57131.13; f.

I.

F o
“”3800 6
-‘~ 5‘ c U‘

85

Table 2.9. PHOSPHATASE ACTIVITIES -— MISCELLANEOUS. A

Sephadex 0100 fraction (from the center of the peak

shown in Figure 2.8) was used.

Substrate (M)

Glucose-l-phosphate
(0.0066)

Glucose-l-phosphate
(0.0066)

Glucose-l-phosphate
(0.066),plus glucose
(0.066)

p-nitrophenyl-phosphate
(0.0066)

p-nitrophenyl-phosphate
(0.033)

Product
Determined

P1
glucose (hexo-
kinase assay)

Pi

p-nitrophenol

p-nitrophenol

Rate

(pmoles/
min/mg protein)

0.215

0.205

0.173
0.0050

0.0068

I M-MNI r—vé-‘J—‘m

L-

. 6
ratios

1'
3.2.1011!

86

products (Table 2.0 and 2.5).

Like the other phosphotransferase activities, al-
most all of the GlP phosphatase activity was found in
the 65-100% ammonium sulfate fraction. In one experi-
ment 8 65-100% ammonium sulfate fraction was chromato-
graphed on Sephadex 0100 and assayed for the FlP,G :
GP and GlP,H20 : G reactions. As seen in Figure 2.3,
the two activities coincided. Chromatography on DEAE
cellulose of Sephadex purified fractions also gave
coincidence of the two activities. As seen in Figure
2.h the phosphatase and hexose phosphotransferase
activities coincided in both cases even though due to
the change in pH there was a shift in the position of
the two activities as we11.as in the overall protein
pattern. Thus it appears that the hexose phospho-
transferase and phosphatase activities are catalyzed

by the same enzyme.

2.2.3. TEMPERATURE DEPENDENT CHANGES IN PHOSPHOTRANS-
FERASE PROPERTIES. During a large scale purification

of gel-filtered H-phosphotransferase fractions on DEAE
cellulose at 0°C, two distinct peaks of FlP,G : GP
activity were found. Repetition of the experiment
starting with a different crude extract gave the same
results. (This particular purification is presented

in Table 2.3.) Chromatography of gel-filtered fractions
at 25°C on a small DEAE cellulose column (Figure 2.5.C)

87

Figure 2.3. COINCIDENCE OF GIP, H20 : 0 AND FlP,G : GP
ACTIVITIES —— GEL FILTRATION. A 1.1.2 m1 aliquot of a
65-100% ammonium sulfate fraction was chromatographed
on a 1000 ml bed of Sephadex 0100 in 0.02 M sodium
phosphate pH 7.5. Assays: (l) GlP,H2O : 0 (specific

glucokinase) (a); (2) FlP,G : GP (0).

88

Figure 2.3.

 

 

 

 

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.02“—

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1
0

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o L.

400 600 800

200

VOLUME (ml)

 

.o
D;

I“nn

d

m 'I-U‘.u_'“. 9"‘FIV-yr—

89

Figure 2.h. COINCIDENCE OF GlP,H2O : G AND FlP,G : GP
ACTIVITIES - DEAE CELLULOSE. A 1.5 m1 aliquot of a

Sephadex purified fraction was absorbed on a 2 ml bed
of DEAE cellulose (0.7 x 2 cm) equilibrated with 0.02
M sodium phosphate, pH 7.5 (A), or pH 6.5 (B), and
was eluted with a 21 ml gradient of from zero to 0.2.
M NaCl in the same buffer. The absorbance at 210 nm
(solid line) was monitored continuously. Assays: (l)
01P,Hg0 : G (hexokinase) 03); (2) FlP,G : GP (0). The
temperature was 25°C; note the incompletely assayed
forward shoulders as in Figure 2.5.C. The gradient
was initiated and terminated at the zero and 20 ml

points respectively.

1 I
'\)

90

Figure 2.4.

 

OHN.Q.O

 

pH 705

I
A.

 

 

 

 

AHA\aHa\mama nufioanv 00.0.mH0

o02 —
0 ~—
.02

AHE\oAa\mZnH uuaoanv 00.0.mHm

 

VOLUME (m1)

 

91

Figure 2.5. TEMPERATURE-DEPENDENT H—PHOSPHOTRANSFERASE
FORMS IN DEAE CELLULOSE CHROMATOGRAPHY. For A and C a
2 ml aliquot of a Sephadex purified fraction was adsorbed
on a 3 m1 bed of DEAE cellulose(0.7 x 2.75 cm) equili-
brated with 0.02 M sodium phosphate, pH 7.5, and eluted
with a 20 m1 gradient of from zero to 0.2 M NaCl in

the same buffer. The column was surrounded by a water
jacket and Chromatography was performed at 00C (A) and
25°C (C). The volume range indicated in C was pooled
and a 2 ml portion was rechromatographed by the same
procedure at 00C (B). The 0.D. at 210 nm (solid'line)
was monitored continuously. Assay: FlP,G : GP (0).
The gradient elution was initiated and terminated at

the zero and 20 m1 points respectively.

Figure 2.5.

FIP.G:GP (uncles TPNH/min/ml)

.03

.02

.01

.01

.03

.02

.01

 

000 _

0.130210

92

 

 

 

 

 

 

 

 

T

l

l l
3 " C. 25°C
21‘
1—

«pooled—o
l
0 10 20

VOLUME (ml)

 

93
gave a single major peak with a forward "tail". At 0°C
(Figure 2.5.A) there were two distinct peaks which
corresponded approximately with the peak and "tail”
observed at 25°C. As determined by the lack of pertur-
bation by the temperature change of the overall protein
patterns,the behavior of the H-phosphotransferase is
not a common property. The ratio of the GlP,H20 : 0
to FlP,G : GP activity was the same, about 5.0, for
both peaks for assays which were made at 25°C. Since
throughout the purification the fractions were kept
at 0-h°C the predominant peak at 25°C could represent
an irreversibly temperature-denatured form. However
when this fraction was rechromatographed at 0°C (Figure
2.5.3) the elution profile assumed the low temperature
distribution pattern indicating a reversible temperature-
dependent change.

The temperature-dependent behavior suggested that
perhaps the phosphatase activity might be modified if
assayed at a lower temperature. However as seen in
Figure 2.6.3 the ratio of the GlP,H20 : G to FlP,G : 0P
activity changed only slightly with temperature varia-
tion, remaining in the same region (about 5) that was
observed using a variety of Sephadex and DEAE cellulose
purified preparations. An Arrhenious plot of the data
gave straight lines'with a downward break at about 2h°C
for both the 01P,H20 : G and FngG : GP activities

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Figure 2.6.
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N H

(eAtquaI) EEVH OTOOI

TEMPERATURE (00)

1 /A°

96
(Figure 2.6.A). A downward break in the Arrhenious plot
indicates a decrease in an activation energy at higher
temperature (61). The apparent heats of activation
(AI-f“) given in Figure 2.6.A are the heats of activa-
tion of the enzyme-substrate complex provided certain
assumptions are made, including the identity of the
rate limiting step. See reference 61 for these assump-
tions. (The AHW values in Figure 2.6.A were calculated
for temperature values of 30°C and 15°C.) The break
in the.Arrhenious plot may be due to the same change
responsible for the temperature-dependent shift in
DEAE cellulose chromatography. The difference in the
temperature range for the two effects could be due to
the difference in the ionic environment, particularly
the difference between adsorption on DEAE cellulose
in one case,and free dissolution in the assays in the
other case. Since the two temperature effects are
fairly independent of each other experimentally, parti-
cularly in that the buffer solutions are different, it
is most likely (61) that there is a direct effect of
temperature on the conformation of the enzyme (as
apposed to an indirect effect due to a temperature-
dependent change of the solvent). A temperature-dependent
change at almost exactly the point at which assays for‘
characterization were made (25°C) adds some uncertainty

as to their general significance.

97

2.2.h. OTHER PROPERTIES. For the experiment shown in
Figure 2.7 a 65-100% ammonium sulfate H-phosphotransferase
fraction was first chromatographed on Sephadex G100 in
0.02 M sodium phosphate, pH 7.5, at 0°C,giving a typical
symmetrical activity peak as assayed with the FlP,G :
GP assay; A pooled fraction was taken,constituting
approximately the center third of the activity peak

(a relatively narrow fraction),and was rechromatographed
on Sephadex 6100 in 0.01 M Tris-HGI, pH 7.0, at 0°C
(Figure 2.7). Assays of the elution profile with the
GlP,HéO : G assay gave an activity peak which, except-
for a slight shoulder or skewed region, did not
correspond with the protein peak. The ratio of GlP,
H20 : GP to FlP,G : GP activity remained at about 5

in both portions of the activity peak. As is generally
found, and in several experiments tried during the
purification of ammonium sulfate fractions of FlPK,
rechromatography of a Sephadex fraction using the same
grade of Sephadex results in a very close coincidence

of the protein and activity peaks. Thus, though it is
far from conclusive, the lack of coincidence of the
protein peak with the activity peak seen in Figure 2.7
may indicate a decrease in the molecular weight of the
Hkphosphotransferase in the absence of phosphate and/or
at pH 7.0. If this were the case the protein peak,

containing a heterogenous mixture of proteins, would

 

,., Menu—

 

98

Figure 2.7. GEL FILTRATION IN TRIS-HCI, pH 7.0. The
sample was a 15 ml aliquot taken from the center third
of a symmetrical peak of FlP,G : GP activity resulting
from the chromatography of a 65-100% ammonium sulfate
fraction on Sephadex G100 in 0.02 M sodium phosphate,
pH 7.5. This was chromatographed on a 200 ml bed of
Sephadex G100 in 0.01 M Tris-HCl pH 7.0. Assays: '(1)
Protein (260-280 nm method) (0); (2) G1P,Hé0 : G (hexo-
kinase) (0).

.H,-O:G (umn‘rnu (I'PNH/min/mi)

II’

(I

99

Figure 2.7.

Aaa\wav szaomm

 

 

o

5
o
_

5
2
a

 

 

 

_
2
o

_

Aaaxnfia\mzmg mofioaav enomm.mHo

100

VOLUME (m1)

100

serve as a marker for the position of the previous higher

molecular weight form. The shoulder in the activity

profile, since it corresponds with the protein peak,
could be due to the higher molecular weight form. In
DEAE cellulose chromatography the H-phosphotransferase
was more quickly eluted (at 29°C) using 0.005 M phosphate
buffen pH 7.5, than with 0.002 M phosphate buffer, pH
7.5. The difference may have been due to the change

in ionic strength. However the position relative to
some of the protein peaks was also somewhat changed.

Gel filtration of a crude extract indicated that

the H-phophotransferase was distinct from some other
phosphotransferases in the extracts (Figure 2.8). in
the high moleculardweight fraction there was a p-nitro-
phenyl-phosphate-reactive phosphatase which may or may
not be responsible for the small peak of GIP phospha-
tase activity in this region. The pH profile of this
ac'tivity is compared with that of the purified H-phos-
Photransferase in Figure 2.9. This activity was
I‘tallowed in the elution profile by the acetyl phosphate,
(5 : G6P activity of the acyl phosphate, hexose phosphate:
hexose phosphotransferase of Kamel and Anderson (6).
'Tlne bulk of the G1P,H20 : c and the FlP,G : GP activity
‘3<:curred in approximately the same region although
'tliere is not an exact coincidence of the two peaks. As

illdicated in Figure 2.8 by the dotted line it is probable
tdnat most of the GlP,H20 : G activity was due to the

 

101

 

Figure 2.8. PHOSPHOTRANSFERASES IN CRUDE EXTRACT OF
FRUCTOSE-GROWN CELLS. An 8 ml sample of a crude
extract of fructose-grown cells (mid-log phase) was
chromatographed on a 200 ml bed of Sephadex G100 in

0.02 M Tris-HCl pH 7.0 plus 0.01 M MgCla. Assays:

 

(l) p—nitrophenyl-phosphate,HQO : p-nitrophenol 03);
(2) acetyl phosphate,G : G6P (.); (3) GlP,H23 : G
(glucose oxidase) 03); (h) FlP,G : GP (0): (S) FlP,

G : GP activity multiplied by 5.0 (dotted line).

102

Figure 2.8.

 

 

200

100

 

 

 

1.0—

5 O
a
o

Suing—\m 32:3 3.4m

VOLUME ( ml)

103

Figure 2.9.PHOSPHATASEACTIVITIBS y§ pH. (A) A Sephadex
G100-purified H-phosphotransferase fraction (from the
activity peak shown in Figure 2.3) was assayed with the
GlP,H20 : Rah assay using 0.1 M sodium acetate (0) or 0.1
M Tris-HCl (0). (B) A crude extract of fructose-grown
cells was assayed for the hydrolysis of p-nitrophenyl—
phosphate as described in Methods, Section 2.u.2.B

using as buffer 0.1 M sodium acetate (0) or 0.1 M Tris—

HCl (Cl) .

 

101;

Figure 2.9.

 

 

 

 

 

2.0+—

?fi 30.3 3}:an 3083 madm

.05 _-

10

pH

1 Che

Figure 2.10. PHDSPHOTRANSFERASES IN CRUDE EXTRACT OF
GLUCOSE-GROWN CELLS. (A) A 10 ml sample of a crude
extract (prepared in 0.02 M Tris-HCl, pH 7.0, plus
0.001 M MgClZ) was chromatographed on a 220 m1 bed of
Sephadex G100 in 0.02 M Tris-HCl, pH 7.0, plus 0.001

M MgClz. Assays: (1) FlP,G : GP (0) [activity multi-
plied by 10 (OU ; (2) GlP,H20 : G (glucose oxidase)
U3); (3) PNH2,G : GP (£9. Fractions from the BA ml

to 132 ml region were pooled. (B) A u ml sample of

the pooled fractions from (A) was adsorbed on a 20 ml
bed of DEAE cellulose (u x 2.5 cm) equilibrated with
0.02 M sodium phosphate, pH 7.5. The column was eluted
with a 200 ml gradient of from O to 0.2 M NaCl in the
same buffer, followed by a 200 ml gradient of from 0.2
M to 0.5 M NaCl in the same buffer, followed by 30 ml
of 0.8 M NaCl in the same buffer. Chromatography was
performed at OéuoC. Assays: (l) FlP,G : GP multiplied

by 10 (-); (2) PNH2,G : GP (Ag.

105

Figure 2.10.

 

 

 

 

 

 

 

 

O-h _

_ P
..Q, 9.
no no

Agi\cas\nuuoeav me<m

0.1

0 200

)5

100

VOLUME (ml)

50

 

 

 

L.4 A Al A
300 uoo

A

A.A.I.A.A

 

 

0.02
0.01 -

:s\53\32§c 932

200

VOLUME (ml)

100

KT.—
'1. ray-aimuv—r‘

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R

‘v

u 9
0

L

.H
l
U.“

105a

H-phosphotransferase if the ratio of GlP,H20 : G to FlP,G

GP activity was the same in this case as it was for purified
fractions. There may also be a second phosphatase with a
slightly lower molecular weight than that of the H-phospho-
transferase. Chromatography of crude extracts on Sephadex
G200, whiCh gives a better resolution in the elution region
involved, results in a substantial shoulder-like tailing

of the GlP,HéO : G activity, suggesting a second enzyme

(V. L. Sapico, unpublished results). Chromatography of a
glucose-grown crude extract on Sephadex G100 (Figure 2.lO.A)
resulted in coincidence of a PNH2,G : GP activity and the
GlP,H§O : GP activity. The peak in the FlP,G : GP activity
preceeded these two activities to a slight extent. In chroma-
tography of fractions from the Sephadex G100 elution profile

on DEAE cellulose at 0-h°C (Figure 2.10.8), both the PNH2
G : GP and FlP,G : GP activities appeared in the two peaks

typical of the H-phosphotransferase. Recovery of the two
activities in DEAE cellulose chromatography was about 50
percent so that it is possible that the lack coincidence

of the FlP,G : GP and PNH2,G : GP activities in Sephadex
chromatography is due to the presence of an enzyme which
does not catalyze FlP,G : GP activity and which is not stable
in DEAE cellulose chromatography. The small shoulder in the
3PNH2,G : GP activity (Figure 2.lO.A) may be due to the

TPNHg-dependent activity catalyzed by the acyl phosphate,

IOSb

hexose phosphate:hexose phosphotransferase of Kamel and
Anderson (6). The significance of the lack of a small
peak of GlP,H20 : G activity in the chromatography of
glucose-grown cells (Figure 2.lO.A) as compared with
chromatography of fructose-grown cells (Figure 2.8)

is not known.

Osmotic shock treatments (Methods) resulted in a
release of the FlP,G : GP activity and an 80 percent
decrease in the rate of acid production by whole cells
from glucose (as determined by the procedure described
in Figure h.h.B). Quantitative results for similar
experiments indicated that at least 60-70% of the H-
phosphotransferase activity could be removed by osmotic

shock treatment (V. L. Sapico, unpublished results).

 

 

i 51‘

b

‘- ll: Adv slit “to
:~ ~ ... 0» cs
as Hit It. t V Oi
. . A c I i A. v C. o
a)... at i Q b u. . ..

..Z'\
I ~‘
I
I‘
h

106

2.3. DISCUSSION

Factors indicating that the H-phosphotransferase
is not involved in the metabolism of fructose specifi-
cally are as follows. (i) The high Km (0.07 M) of‘the
enzyme for fructose. (ii) The high level of phospha-
tase activity catalyzed by the enzyme and its lack of
specificity for reactions catalyzed. (iii) The lack
of inducibility of the enzyme. (iv) The establishment
of a phosphoenolpyruvate-dependent phosphotransferase
as the source of FlP by both.1g‘ggtgg data and muta-
tional analysis. (v) The inability to implicate the
H-phosphotransferase specifically in the metabolism of
fructOse by mutational analysis (Section h.2.3). It is,
however,likely that the enzyme has a role in the meta-
bolism of carbohydrates, hexoses in particular. The
present investigation is for the most part unsuitable
for determining what this role might be since it was
primarily oriented only toward fructose metabolism.
However some general suggestions and relationships with

respect to similar enzymes can be made.

Two classes of phosphotransferases to which this
enzyme is related have been isolated from bacteria; the
sugar phosphate phosphohydrolases and the hexose
jphosphate:hexose phosphotransferases. The sugar phos-

:phate phosphohydrolases are a subgroup of the bacterial

 

 

.nnu. -.LII‘I

 

 

so

107

"acid phosphatases", a term which is used for any phos-
phatase with a pH optimum in the acidic or neutral
range (62). The acid phosphatases include a wide variety
of enzymes which are nonspecific and a number which are
specific for relatively uncommon substrates. The
results of a number of investigations (63-67) have pro-
duced what appears to be two major categories of
bacterial acid phosphatases. One class is very active
on nonbiological phosphates such as nitrophenyl-phos-
phates. This class may be composed of a very hetero-
geneous and unrelated group of enzymes. The nitro-
phenol-phosphate phosphatase activity found in the
chromatography of a crude extract (Figure 2.8) may be
catalyzed by this type of enzyme(s). The second class,
the sugar phosphate phosphohydrolases, is active pri-
marily on sugar phosphates, particularly hexose phos-
phates, but also in many cases phosphates of pentoses,
disaccharides and hexose derivatives. The activity of
these enzymes with nitrophenyl-phosphates varies from
zero to 100% of the activitwaith other substrates.

As suggested by Rogers and Reuthal (63) there appears
to be a gradient in the extent of nitrophenyl-phosphate
phosphatase activity which is catalyzed by the different
sugar phosphate phosphohydrolases of E. 9311, The best
characterized sugar phosphate phosphohydrolase is that
from figisseria meningitidis (6S). Enzymes representa-

tive of both of the two major classes of bacterial acid

108

phosphatases have been found to be removed by osmotic
shock treatments (6h, 65, 66, 68). In g.‘ggli at least
one of the sugar phosphate phosphohydrolases is induced by
growth on gluconeogenic substrates (69) [perhaps that
one which is most active on FDP and 2,3-diphosphoglycerate
( 66EL Wide variations in sugar phosphate phosphohydrolase
levels occur in g. 521; under different growth conditions
and at different phases of growth (6h, 66, 69, 70).

Several types of hexose phosphate:hexose phosphoe
transferases have been isolated from bacteria. (i) Smith
33 a; (71-75) have isolated an enzyme from g. 22;; which
catalyzes the reactions:

(a) PNH2 (phosphoramidate) + Glucose-———é-G1P

or G6? + NH:
(b) G1? + Glucose$===> Glucose + G6P
(c) PNH2 + HéO-——-= Pi + NH3
(d) GlP or G6P + HéO-———=~Glucose + P1

Previously, some of these reactions were thought to be
catalyzed by separate enzymes since occasionally the
activities were separated in chromatography and varia-
tions in activity ratios were observed in a variety of
conditions (73-75). The most recently reported form

of the enzyme (71, 72), which is purified almost to homo-
geneity, gives rates for reactions (b) and (d) of about
25 percent those of (a) and (c). The Km for glucose is

about 0.01 M’and other related compounds also serve as

(95'
M
cast

ll « -. Pd 0,.
1 . Z .0.
A v 7.. c

a. My. .ot Wu

..Ilintrh 1.....‘1
ll-’o. h . . r .

ther

 

109
acceptors (73). Catalysis via a phosphorylated enzyme
has been implicated (72). (ii) Smith 3; §l_(76) have
also found in Mygobacterium.smegmantis an enzyme which
catalyzes reaction (a) and which may or may not catalyze
reaction (c). [A different enzyme in the same organism
catalyzes reaction (b) but not (d) (76 ).] The Km for
glucose of this enzyme, like that of the enzyme in E.
gall, is about 0.01 M"which is rather high. This
enzyme is also active‘with FlP as a donor but appears
to make only F6P from GlP and fructose. In one early
experiment (7h) the PNHZ-active enzyme from E.‘gg;i
made primarily F1? from fructosefplus PNHZ. However
there has been some uncertainty as to the product com-
positions produced by this enzyme. (iii) The acyl phos-
phate, hexose phosphate:hexose phosphotransferase
isolated by Kamel and.Anderson (6) from A. aerggenes
appears to be distinct from.the enzymes investigated
by Smith 23'21, The Km for glucose (1.6 x 10.."L M) is
well‘within the ig‘zigg range. Km values for other
sugars are marginally inside (Km for mannose is 0.01 M)
or outside of the expected in 3133 range (Km for fructose
is 0.3 M). This enzyme, like the PNHZ-active enzyme
from E'.22£1 catalyzes phosphatase reactions (at about
the same rates as hexose phosphorylations) which are
nmrkedly inhibited in the presence of a suitable acceptor.

In an investigation directed toward phosphatases the

 

I:

.¥

K».

 

110
H-phosphotransferase would have appeared to have been a
major sugar phosphate phosphohydrolase of A, aerogenes.
As such, the enzyme is exceptional in its lack of re-
activity with FDP which is hydrolyzed at maximal rates
by all forms of the sugar phosphate phosphohydrolases
in E. ggli.(63) and that in E. meningitidis (65). A
wider survey of substrate specificity might indicate
that activity is almost completely restricted to glucose
and fructose monophosphates. The pH optimum and extract-
ability by osmotic shock treatment are also typical of
sugar phosphate phosphohydrolases. However in those
areas in which the data overlap, the H-phosphotransferase
appears to be the analogue of the PNHa-active enzyme in
E. gall (71-75) except that the present enzyme has a
greater affinity for glucose. It also has a considerably
higher ratio of phosphatase to hexose phosphotransferase
activity than the PNH2-active enzyme in E. 2311, However,
as for the other phosphotransferases described, the affinity
of the H-phosphotransferase for glucose places it in terms of
kinetics in the hexose phosphotransferase class rather than
the phosphatase class, regardless of the relative rates of the
reactions catalyzed. The actual identity of the enzyme de—

pends of course on its 13 vivo role. The finding that the

111

H-phosphotransferase is apparently responsible for a
substantial proportion of the sugar phosphate phospho-
hydrolase activity which can be purified from a crude
extract (Figure 2.8), and the relatively high rate of
the hydrolytic reactions catalyzed by the enzyme,
suggest that, at least gaugiggg, there may not be a
well defined distinction between some of the presently
known bacterial sugar phosphate phosphohydrolases and
sugar phosphotransferases. Insofar as we are aware
none of the bacterial enzymes presently characterized
as sugar phosphate phosphohydrolases, and only one
bacterial phosphatase of any type, has been tested for
phosphorylation of substrates (62). It is possible
that some of these enzymes including the H-phosphotrans-
ferase are 13 11559 forms of enzymes which do not
catalyze phosphatase reactions i3 yivg. For those

associated with the membrane, in vitro conditions may

 

be particularly disrupting of normal function. This
possibility has been suggested for the acyl phosphate
hexose phosphate:hexose phosphotransferase in.A,

aerogenes (6). From mutational evidence (Section h.2.3)

 

it does not appear that the H-phosphotransferase is a
degraded form of the Enzyme II component of the phospho-
enolpyruvate-dependent system described in Section 3.
Fluoride inhibition and removal by osmotic shock are

also not exhibited by Enzyme II components in other

 

o .

ac-

lllll!..'..or§..al1drn 5‘
o e

law... ; 4 -t

 

 

112

organisms (77). Such a relationship also seems to have been
ruled out for the PNHg-active enzyme in E, 33;; (72).

very recently Schaefler and Schenkein (78) have provided
details on a third hexose phosphate:hexose phosphotransferase
from A, aerogenes. The enzyme also catalyzes a somewhat
atypical set of sugar phosphate phosphohydrolase reactions.
This enzyme is distinguished from the H-phosphotransferase
by its hydrolysis of p-nitrophenyl-phosphate and FDP, and
its lack of activity with GlP. The kinetics of the enzyme
were complex and there was an apparent change in a phospha—
tase to hexose phosphotransferase ratio over a broad range
of temperature. In some instances the activity was re-
solved into a double peak by acylamide gel electrophoresis.
The reason for this was not established. It was proposed
that a conformational change dependent on the aromatic ring
of p-nitrophenyl-phosphate was responsible for certain
kinetic effects. Smith E£.2l (71, 76) have also reported
the presence of a phosphotransferase of unspecified type

in A, aerogenes, quite possibly the same enzyme as the H-

 

phosphotransferase, although we are unfamiliar with the de-
tails (79). Before there is any possibility of under-
standing the phosphotransferases in A, aerogenes it would
appear that an integrated investigation will be necessary

in which all of the phosphatase and other phosphotransferase
activities under a variety of growth conditions would be

correlated with one another.

113

For none of the enzymes described above has it been

possible to establish lg’vivo roles. (See reference 80

 

for a review and critique of metabolic roles suggested
for acid phosphatases.) The establishment of the £2
33!3_roles for these phosphotransferases requires the
isolation of mutants lacking them but apparently in no
case has this been possible (6h). However Englesberg
gt al_(81) have investigated a class of glucose-
resistant mutants,having a 2 to 3-fold elevation of

hexose phosphate phosphatase,which show a perturbation

in 32P‘uptake suggesting that 12_ylyg the phosphatase
releases free phosphate from phosphate esters. The
elevation of acid phosphatase in this series of mutants
was closely associated with defective glucose permeation.
An increase in sugar phosphate phosphohydrolase activity
has also been noted in E. 99;; grown on limiting concen-
trations of glucose (70), and it has been suggested (63)
that the enzyme is involved in the production of intra-
cellular hexoses either via transport or by hydrolysis
of hexose-phosphates. A sugar phosphate phosphohydrolase
in E‘.23l1 (perhaps the same one as above) was found to
be repressed in the presence of hexoses and induced
during growth on acetate, alanine, ribose and gluconate
(69). As suggested by these results it was thought that
perhaps the H-phosphotransferase might produce free

glucose from glucose phosphates to supply an unknown

1111

biosynthetic reaction so that mutants lacking it would
gr ow only on glucose. Although one such mutant lacking
H-phosphotransferase activity was isolated it is doubt-

ful that this hypothesis is supported (Section 11.2.3).

 

 

to.

115

2.11. METHODS

The preparation of crude extracts and other procedures

not mentioned here were as described in Section l.h.

2.h.1. PURIFICATION OF THE H-PHOSPHOTRANSFERASE. For the
purification outlined in Table 2.3 a crude extract of
fructose-grown cells was treated with protamine sulfate
(Section l.h.3.A) and 0-66% and 66-100% ammonium sulfate
fractionS‘were made (Section l.h.3.A). A 25.5 ml aliquot
of the 66-100% ammonium sulfate fraction was chromatographed
on a 1,000 m1 bed of Sephadex G100 in 0.02 M sodium phos-
phate buffer, pH 7.5. A 50 ml aliquot of the center
fractions from Sephadex chromatography were adsorbed on a
100 m1 bed of DEAE cellulose (5.5 x h.7 cm) equilibrated
eta-11°C with 0.02 M sodium phosphate, pH 7.5. The column
was eluted‘with a 1,600 m1 gradient of from zero to 0.2 M
NaCl in the same buffer,giving two activity peaks similar
to those in Figure 2.5.A of which only the slowiy chroma-
tographing peak was pooled. The FlP,G : GP assay was used
throughout the purification. The loss of activity during
ammonium sulfate fractionation was the only actual loss
observed. In Sephadex and DEAE cellulose chromatography
‘the enzyme was completely stable, the loss of activity
‘being due to discarded fractions. Sephadex and DEAE

(sellulose fractions were stable for up to 3 months stored

a t 041°C

 

 

116

2.11.2. ASSAYS.

A. Enzymatic Assays. Numbers are pmoles per 0.15 ml

total volume containing 10 umoles glycylgylcine, pH 7.5,
and components are listed in the order of addition. See
Section 1.11.2.A for general techniques for enzymatic assays.

l. GIP,H20 : G (glucose oxidase) - The assay was identical

 

to the sucrase (glucose oxidase) assay (Section 1.11.2.C),
minus sucrose plus GlP 1.0; blank-minusextract.

2. GlP,H20 : G (hexokinase or specific glucokinase)- TPN
0. l , glucose-6-phosphate dehydrogenase, yeast hexokinase
or specific glucokinase purified from A. aerogenes, Mng2
2.0, ATP 1.0, extract, GlP 1.0; blank-minus ATP. This
assay could not be used with crude extracts due to inter-
ference by phosphoglucomutase.

3. FlP,G : GP - This assay was used routinely for the
Phosphotransferase. TPN 1.0, glucose-6-phosphate dehydro—
genase, MgC12 1.0, phosphoglucomutase, extract, glucose
100, FlP 1.0; blank — none appreciable using 0.01 ml of
crude extract. ,

11° GlP,F : FlP - DPNH 0.07, tx-glycerophosphate dehydrogenase
plus triose phosphate isomerase, aldolase, MgCl2 1.0, ATP
0'5: FIPK 0.02 ml (calcium phosphate gel purified, con-
taining’0.l M sodium phosphate), extract, fructose 10.0,
GIP 1.0: blank-minus fructose (a high blank value was ob-

tained using crude extracts).

117

5. FlP,G : G6P - Same as FlP,G : GP, minus phosphogluco-

 

mutase: blank-minus glucose.

6. F6P,G : GP - TPN 0.1, glucose-6-phosphate dehydro-

 

genase, MgC12 1.0, phosphoglucomutase, extract, glucose
1.0, F6P 0.h: blank-minus glucose (preparation must be
relatively free of phosphoglucose isomerase).

7. F6P,G : G6P - Same as F6P,G : GP, minus phosphogluco-

 

mutase; blank-minus glucose.

 

8. GlP,G : GP - Same as FlP,G : GP, minus FlP, plus GlP

 

1.0; blank-minus glucose.

9. FlP,F : F6P - TPN 0.1, glucose-6-phosphate dehydro—

 

genase, phosphoglucose isomerase, MgC12 0.2, extract,
fructose 10.0, FlP 1.0; blank-none appreciable using 0.01
ml of crude extract.

10. G6P,F : F1P - Same as GlP,F : FlP, minus G6P, plus

 

GlP 1.0; blank-minus fructose.

11. GlP,F : F6P - Same as FlP,F : F6P, minus F1P,vplus

 

GlP 1.0; blank-minus fructose.

l2. Acetyl phosphate, G : G6P - TPN 0.1, glucose-6-phos-
phate dehydrogenase, MgC12 0.5, extract, glucose 1.0,
acetyl phosphate 0.5.

13. p-Nitrophenyl-phosphate, H20:p-nitrophenol-MgC12 0.5,
extract, p-nitrophenyl-phosphate 1.0; 0.D. at 1120 nm, 0.77

icrxo.h20/o.01 pmole/o.15 ml; blank-none appreciable.

llI-III'?‘ .

)l'l‘o‘oovoi.lou.. 9|. .lflofi
, ' p

q.

9”

v f

 

118

lb. Survey Assay for FlP Formation - Same as the GlP,F :
FlP assay, minus GlP, plus phosphate donors at the concen-
trations given in Section 3.2.1. The FlPK preparation was

a Sephadex purified fraction.
15. PNH2,G : GP - Same as FlP,G : GP, minus FlP, plus phos—

 

phoramidate 1.0: blank-minus glucose.
B. Other assays.

Phosphate formation from sugar phosphates - The re-
action mixture contained in a total volume of 0.1 ml: 1
pmole Tris-HCI, pH 7.5 (unless noted otherwise), 1.0 umole
sugar phosphate and enzyme in 0.01 M Tris-HCl, pH 7.5. In-
cubations were made for zero, 20, ho, 60 and 100 minutes
and the zero values were taken as the blank due to free
phosphate in the enzyme preparation and sugar phosphate.
The reaction was terminated by addition of aliquots to
the SubbaRow phosphate assay components (h8).

p—Nitrophenyl-phosphate hydrolysis at different pH

values (Figure 2.9). The assay contained in a volume of

 

0.h m1: ho pmoles of Tris-HCl or sodium acetate (at the
indicated pH, Figure 2.9), 5 pmoles p-nitrophenyl-phosphate
and crude extract. After zero to 6 minutes the reaction
was terminated and the color was developed by the addition
of 0.h m1 of 1 M Tris-HCI, pH 8.5, containing 0.h M
potassium phosphate and the O.D.u20 was determined (1 cm
light path), o.77A0.o./o.01 pmole/0.15 m1).

119

2.h.3. .ATTEMPTS TO ISOLATE H-PHOSPHOTRANSFERASE PRODUCTS.
Two attempts were made to isolate sugar phosphate products
from reaction mixtures containing purified H-phosphotrans-
ferase. Experiment No. 1 - The reaction mixture containing
in a total volume of 5.25 ml, 1.0 m1 of a 60-100% ammonium
sulfate fraction (similar to that in Table 2.3), 500 pmoles
fructose, 100 pmoles GlP and 50 pmoles MgC12,was incubated
at 25°C for 21 minutes and heated at-lOOOC to stop
the reaction. The pH during incubation was maintained at
7.5 by titration with dilute NaOH. Chromatography of the
reaction mixture on a Dowex 1-borate column similar to
that used for preparation of the product of FlPK (Section
l.h.h) resulted in only a single peak in the free sugar
region. Experiment No. 2 - The reaction mixtures con-
tained (in a total volume of 0.6 m1),0.09 pmole/min of
DEAE cellulose purified H-phosphotransferase and 20 pmoles
sodium phosphate, pH 6.7, plus (in pmoles per total volume):
(a) FlP 3.5, glucose 3.5; (b) GlP 3.5, fructose 7.0; (c)
FlP 3.5, glucose 3.5, MgC12 3.h; (d) GlP 3.5. The mixtures
were incubated at 25°C for one hour, heated at 100°C,
treated with Dowex 50 to remove Mg++ and chromatographed
on paper eluted with water saturated phenol, and developed
with silver nitrate (56). In all cases the free sugars
were the major components in the reaction mixtures with

only traces of sugar phosphates detectable.

120

2.11.11. OSNDTIC SHOCK TREATMENT. The procedure is one of
those described by Rbssal and Heppel (68). Cells grown
overnight on fructose were harvested by centrifugation at
25°C and washed once at 25°C with 0.03 M Tris-HCI, pH 7.5
(250 ml/g wet wt). Cells were suspended at 25°C in 0.03

M Tris-HCl, pH 7.5, containing 20% sucrose, 0.001 M EDTA
and 0.001 M MgC12. The suspension was incubated at 32°C
for 10 minutes with agitation and the cells were harvested
by centrifugation at 25°C. The cells were then very
rapidly suspended in ice cold 0.001 M'MgCl2 (100 ml/g wet
wt) using a large syringe to break up the pellet. Cells
'were removed by centrifugation and the supernatant was
used for enzyme assays. The cells were suspended in

0.85% NaCl and acid production was determined as described
in Section h.3.3 using an incubation mixture containing
0.85% NaCl.

2.h.5. REAGENTS. Those not mentioned here are given in
Section l.h.5. Specific glucokinase froqu. aerggenes
(about 200-fold purified) was the gift of R. L. Palmer.
Hexokinase (yeast) and phosphoglucomutase (yeast) were
from Calbiochem. F6P was purified by chromatography on a
Dowex-l-borate column using a procedure similar to that
used in Section l.h.h. Other compounds were: GlP (N.B.C.),
G6P (N.B.C.), acetylphosphate, dilithium salt (Calbiochem),
carbamyl phosphate (Sigma), mannose-6-phosphate.(N.B.C.),
ribose-S-phosphate (Sigma), phosphocreatine (Sigma),

121

D—3-phosphoglycerate (Sigma), inosine triphosphate (Pabst),
DL-«-glycera1dehyde-3-phosphate (Sigma) and p-nitrophenyl-
phosphate (Sigma). Sodium salts of the above compounds
were used unless noted otherwise. Potassium phosphorami-

date was prepared from diphenylphosphoramidate (73).

SECTION 3. PHOSPHOENOLPYRUVATE-DBPENDBNT
FORMATION OF FRUCTOSE-l-PHOSPHATE BY
A FOUR-COMPONENT PHOSPHOTRANSFERASE SYSTEM

SUMMARY: In this section we show that in A. aerogenes
fructose is phosphorylated with phosphoenolpyruvate
specifically at carbon atom l by a phosphotransferase
system of the type originally described by Kundig A;

El (82) for other sugars. The phosphotransferase

system for fructose has been resolved into four com—
ponents: enzyme I, HPr and two components required

for enzyme II activity. The enzyme II components are

a liigh molecular weight protein and an inducible protein
of .lower molecular weight, which is proposed to be the

dettarminant of sugar specificity for a multicomponent

enzyme I I system.

3.1. INTRODUCTION

The phosphoenolpyruvate-dependent phosphotrans-
feraise system (PEP system) discovered by Kundig g;
11 111 E. 22A; involves the three components HPr,
enz3nne I and enzyme II in the two reactions:
(1) Enzyme I

PEP + HPr E==§ Pyruvate + phospho-HPr
(2) Enzyme 11

Sugar + phospho-HPr-———A Sugar phOSphate + HPr

122

'r r. v' c -. run-n ———o

i
'1
j

 

 

123

The sugar specificity of the system is determined by a
series of enzymes II which are high molecular weight mem-
brane-bound enzymes. PEP systems have subsequently been
found in other bacteria and have been implicated in the
transport of sugars across the cytoplasmic membrane as
‘well as in their phosphorylation (see references 83 and
8h for reviews).

Evidence which suggested that a PEP system is the
source of FlP in A, aerogenes was as follows. (i) As
described in Section 2 the hexose phosphate:hexose
(H20) phosphotransferase was found to have several £2
31532 properties suggesting that it is not a suitable
source of FlP‘lg‘zggg. (ii).As described in Section
11.2.5 it was not possible by mutational analysis to estab-
lish an igigggg role in fructose metabolism for this phos-
photransferase. (iii) As described in Section R a class
of mutants was isolated which had the hexose phosphate:
hexose (H20) phosphotransferase activity as well as FIPK
but which showed impaired growth Specifically on fructose.
(iv) Tanaka and Lin (85) had isolated a mannitol-negative
mutant of A, aerogenes which lacked an enzyme 11 activity
for mannitol. This was the first instance in which a
carbohydrate-negative mutant was found to involve a PEP
‘system component. This provided convincing evidence
that PEP systems‘were a suitable answer to the "missing

kinase problem" (86). Further, during the course of this

1211

work Tanaka and Lin (87) made the extremely important dis-
covery that a class of pleiotropic mutants of A, aerogenes
involved the loss of either enzyme I or HPr. With the excep~
tion of the work of Egan and Nbrse (88, 89, 90), up to this
point pleiotropic carbohydrate-negative mutants had been
almost ignored in work on bacterial carbohydrate metab-
olism even though they are probably the most common type
of mutants isolated (91). The enzyme I and HPr negative
mutants isolated by Tanaka and Lin (87) showed impaired
metabolism of mannitol, sorbitol, glucose, mannose,
fructose and glycerol with some differences in growth
rates between the two types of mutants on these substrates.
Since by analysis of an FDPase-negative mutant (92) as
‘well as FlPKanegative mutants (Section h.2.h) it had been
shown that the FlP pathway is the primary route of fruc-
tose metabolism in A, aerogenes it appeared almost cer-
tain that there must be a PEP system in A, aerogenes
which produced FlP, although all of the PEP systems inves-
tigated up to this point were thought to form hexose-6-
phosphates specifically (93).

A PEP system which forms FlP specifically and which
has properties indicating that it is the 13 3313 source
of FIP was found. It was also found that the enzyme 11
activity for fructose involves a two-component system
with properties which may be applicable to other enzyme
11 activities. A model (Section 3.3.2, Figure 3.2M) for

125
this enzyme 11 system is proposed.
Portions of this section are covered in references 9h

and 95.
3.2. RESULTS

3.2.1. UNSUCCESSFULINITIAL.ATTEMPTS TO PURIFY PEP SYSTEM
COMPONENTS. As will be described, a low level of a PEP-
dependent formation of FlP was detectable in incubations
of crude extracts of fructose- and glucose-grown cells
using an enzymatic end-point assay for FlP. Attempts
were made to separate this system into the three expected
components (HPr and enzymes 1 andII) by chromatographing
crude extracts of fructose- and glucose-grown cells on
Sephadex G200 and assaying recombined fractions with the
FlP end-point assay. Using this method the general out-
lines of a three-peak system were evident. However the
results were not definitive because the end-point assay
was so insensitive and time consuming that most of the
activity of the fractions was lost before the assays
could be completed and because, as is now known, the
system.is unusually complex. It was also evident that
purification beyond the gel filtration stage would be
impractical using the end-point assay due to dilution of
the preparations. Thus it was necessary to devise a
continuous spectrophotometric assay for at least enzyme

I and HPr.

‘.’o_

 

IT.

11.1 ' .

 

19.0“.

(a
aw.-
1

126

An attempt was made to use a continuous glucose-6-
phosphate dehydrogenase-linked assay for PEP dependent
production of glucose-6-phosphate. Analysis of gel
filtration elution profiles of extracts of glucose-grown
cells gave the expected three peaks when fractions were
recombined. However upon closer examination it was
found that PEP could be replaced by.ATP with no loss of
activity of gel-filtered components and that the three
peaks corresponded (at least in several experiments)
to pyruvate kinase (high M,W.),glucokinase (intermediate
M.W.) and.ADP (low M;W.) so that the reaction catalyzed
may have been:

(1) Pyruvate kinase
PEP + ADP —->- Pyruvate 4- ATP
(2) Glucokinase

ATP 4- Glucose ———\ G-6-P + ADP
Thus it appeared that though a PEP system for glucose
may have been resolved in some of the experiments and
could probably be purified if special precautions were
taken, a kinase producing the same product as the PEP
system would produce a continual source of ambiguity

during purification.

3.2.2. .A CONTINUOUS SPECTROPHOTOMETRIC.ASSAY'FOR.A ONE-
STEP RESOLUTION OF PEP SYSTEM COMPONENTS. Tanaka and
Lin have shown that there is a inducible PEP system for
mannitol in A, aerogenes (85). Since there is no

127
ATP-dependent kinase for mannitol in A, erogenes (7) it
was possible to develop a continuous spectrophotometric
assay for the components of the mannitol PEP system. As
will be discussed below, the assay involved the reaction
sequence:
(1) PEP system

PEP + Mannitol ———>-annitol-l-phosphate + Pyruvate

(2) Mannitol-l-phosphate dehydrogenase
Mannitol-l-phosphate -+ DPN+;==‘- Fructose-6-phosphate
+ DPNH

(3) DPNH oxidase
DPNH + 02 —> DPN+ + H202

(h) G1ucose-6-phosphate isomerase
Fructoseé6-phosphate ;::2:Glucose-6-phosphate

(5) G1ucose-6-phosphate dehydrogenase
Glucose-6-phosphate + TPN+R;::i 6-Phosphogluconolac-
tone + TPNH

The mannitol-l-phosphate dehydrogenase preparation used

was purified from mannitol-grown A, aerogenes (Methods,

Section 3.h.5).

Chromatography of crude extracts of mannitol-grown
cells on Sephadex G200 and recombination of fractions gave
an elution profile with three peaks which, on the basis of
expected molecular weight values as observed in g. ggii (82,
93), correSpond with enzyme 11 for mannitol, enzyme 1 and

HPr as labeled in Figure 3.1. The HPr fraction was

 

 

 

 

128

Figure 3.1. GEL FILTRATION ELUTION PROFILE OF A CRUDE
EXTRACT OF MANNITOL—GROWN CELLS. A 65 m1 sample of
crude extract, prepared without mercaptoethanol, was
chromatographed on a 650 ml bed of Sephadex G200 in
0.0h M Tris-HCl, pH 7.6, without mercaptoethanol.
Assay components: Enz II Mtl (gf 1X) was a center
fraction of an enzyme II peak obtained in a similar
gel filtration experiment and was stored at 0°C for

6 days in the absence of mercaptoethanol before use.
Enz I Mtl (gf 1X) was from the h20-h30 ml fraction
of this profile. HPr Mtl (gf 1X) was from the 520-
530 m1 fraction of this profile. ,Assay compositions:
(1) For enzyme II (0) — mannitol assay, 0.02 ml Enz I
Mtl (gf 1X), 0.02 ml HPr Mtl (gf 1X), with ATP. (2)
For enzyme I (A) - mannitol assay, 0.005 ml Enz II
Mtl (gf 1X), 0.02 ml HPr Mtl (gf 1X), without ATP.
(3) For HPr (D) - mannitol assay, 0.005 ml Enz II

Mtl (gf 1X), 0.02 ml Enz I Mtl (gf 1X), without ATP.
(h) Conductivity measurements (0) are in arbitrary
units and the value for the elution buffer has been

subtracted.

oom Aaav mammo>
. oom co:
cow

 

   

spasaposaaoo

129

 

Hum HH Nsm\\\l.

 

FigueBJ.

 

I “Go

 

(Tm/UTm/setomfi) aava

130

completely stable to heat treatment for 10 minutes at 100°C.
It'was not located in the lowest molecular weight fraction
of the gel filtration profile as ADP would be. This was
indicated by the observation that the peak in electrical
conductivity did not coincide‘with HPr (Figure 3.1). The
increase in conductivity shown in Figure 3.1 was due to

an increase in buffer concentration at the void volume
since the sample contained about 0.075 M buffer whereas
0.0h M buffer was used for elution. Chromatography of HPr
samples from Sephadex G200 chromatography on Sephadex G25
also gave activity peaks in the higher molecular weight
region rather than at the void volume. The HPr activity
ems completely retained during repeated ultrafiltration
through a membrane with a 10,000 M;w. cut-off value. The
heaviest peak, the Enz II Mtl (gf) fraction (see Methods,
Section 3.h.2.A for terminology) as indicated by comparison
of Figure 3.13A and Figure 3.13B, was induced by mannitol,
eliminating the possibility that it was pyruvate kinase
and consistent with the expected properties of an enzyme
II for an inducible PEP system. Thus by elimination and
as indicated by its constitutivity and relative molecular
weight the middle peak may be designated enzyme 1.

Enzyme I retained activity for only a few days in the
absence of mercaptoethanol. The low level of enzyme I

in Figure 3.1 as compared with other profiles was probably

due to the absence of sulfhydryl protection in this

131
experiment. In the presence of mercaptoethanol enzyme 1
retained useful activity for only about two weeks stored
at 0°C thus making its availability the limiting factor
in purification of PEP system components. Enzyme II
and HPr were stable for at least four to eight weeks
stored at 0°C in the presence of mercaptoethanol. The
three components from gel filtration were purified further
on DEAE cellulose by stepwise elution as shown in Figure
3.2. The double peaks may be due to the discontinuity

of the elution procedure.

3.2.3. REQUIREMENTS OF THE ASSAY FOR THE MANNITOL PEP
SYSTEM DURING PURIFICATION. Tables 3.1, 3.2 and 3.3

give the requirements of the mannitol assay throughout

the three stages of purification. With the exception

of ATP which will be considered below, the expected

pattern of increasing requirements with increasing puri-
fication was observed. The requirement for mannitol-1-
phosphate dehydrogenase which was achieved (partially) after
DEAE cellulose purification and the DPN requirement of the
gel filtration purified components are consistent with
identification of the product as mannitol-l-phosphate
(which is identical to mannitol-6-phosphate). Since
lnannitol-l-phOSphate dehydrogenase is an inducible enzyme,
assays of gel-filtered enzyme 1 and HPr fractions from
extracts of fructose-grown cells required added mannitol-1-

phosphate dehydrogenase to a greater extent than assays

l‘
. .31:
,fi

, i .177 ”my"-

m‘.‘
1

(A2;

132

Figure 3.2. DEAE CELLULOSE ELUTION PROFILES OF PEP SYSTEM
COMPONENTS. A 20 m1 bed of DEAE cellulose in a h x 2.5 cm
column was eluted with aliquots of 0.0M M Tris-HCl,

pH 7.6, containing 0.2% mercaptoethanol and increasing
concentrations of NaCl. Molarity figures indicate the
approximate point at which a step-up to that concentra-
tion occurs. The figures are aligned so as to give
correspondence of molarities in certain regions.

Samples and recovery of activity: (A) h ml of Enz I

Mtl (gf 1X): h5% recovery. (B) h m1 of HPr Mtl (gf

1X); 110% recovery. (C) 2 ml of Enz II Mtl (gf 1X);

110% recovery. (D) 2 ml of Enz II F (gf 1X); 75%
recovery. Assays for A, B and C were the same as

those described in Figure 3.1 except that ATP was used in
all cases. For D the fructose assay (Methods) was used
containing per 0.5 ml incubation mixture: 0.05 ml

sample, 0.05 ml Enz I F (gf 1X) and 0.05 ml HPr F

(gf 1X).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

133
Figure 3.2.
'a‘
‘\ l I I
5 A. Enz I Mtl
E
gig o01"— _
at:
(0
(D
'6' onnnnndxnr‘ndnn l l nnnnn
E 0 0.1 0.2 0.3 0.4
I |
NaCl M
0 ml ( ) 100 ml
H
E I I
n B HPr Mtl
H O
S
as
B
001'— .—
3181
(I)
(D
'3
B 0 rxn n A n d n n n n m n n n A n n
1
O 02. 0.4 0.8
l
0 ml NaCl (M) 100 m1
\.
R I l l I
13133 C. Enz II Mtl d
ans ~005-
<1a>--I
mgfi 0 lnAn/xntk Arlnn
{—3, o 0.1 o. 2 0.5
| I
0 ml Nam (M) 80 ml
E f l l I f l
m.4 '01 D. Enz II F "
823%
was
a 0 I an n I n n I l
O 0.1 0.2 0.3 0.4 0.5

' NaCl (M) I
0 ml 60 m1

alga-

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138

of components from extracts of mannitol-grown cells. TPN
‘was required for both the crude extract and gel-filtered
components. The absence of DPNH production,as indicated
by a lack of activity in the absence of TPN,is to be
expected since the DPNH which is produced would be imme-
diately oxidized by the high levels of DPNH oxidase
present in these fractions. If the incubations were
continued for a long enough time, at a certain point,
depending on the initial rate of the reaction, a sharp
break occurred and the rate doubled. Stirring the
cuvettes caused resumption of the normal rate. Observa-
tions of the glucose oxidase (Glucostat) assay, involving
an oxygen requiring reaction which produces a visible
color, indicated that in the small'narrow cuvettes used,
the dissolved oxygen was exhausted in the bottom of the
cuvette much faster than it could diffuse in from the top
layer. Thus for the mannitol assay,since the light path is
confined to the bottom layer, long incubation results in
exhaustion of the oxygen supply for the DPNH oxidase re-
action so that DPNH as well as TPNH are produced, causing
an increase in rate. A similar situation was noted in
attempts to assay in crude extracts the DPNH-producing
enzyme, glyceraldehyde-3ephosphate dehydrogenase, except
that in this case there was a rapid enough formation of
iDPNH relative to its utilization by DPNH oxidase to produce

a detectable increase in 0.D. even in the presence of

139

dissolved oxygen. The 0.D. very quickly reached a plateau
value, presumably due to the establishment of a steady
state level of DPNH at that point on the DPNH saturation
curve of DPNH oxidase which allowed a rate of oxidation
equal to that of reduction. These observations are perti-
nent to the explanation for the ATP effect described below.
The ATP dependence seen in Table 3.1 and 3.2 is a
requirement for the assay system but not of the PEP system
itself. using crude extracts of mannitol—grown cells and
using either ATP or PEP alone, there was a lag period of
about 10 minutes before the rate assumed a maximum value.
In the presence of both.ATP and PEP there was no lag
period. This indicates that perhaps both.ATP and PEP
are required for the assay and that in crude extracts
they are interconvertible by various enzymes (pyruvate
kinase for instance) which require low molecular weight
endogenous compounds (such as ADP and pyruvate). The a1~
most complete requirement of gel-filtered components for
ATP as well as PEP is consistent with the involvement of
endogenous compounds, which are removed by Sephadex chromato-
graphy, in the interconvertibility of PEP and ATP in crude
extracts. Surprisingly however purification of the enzyme
11 fraction on DEAE cellulose caused a loss of the.ATP re-

quirement (Table 3.3). The explanation for this appears to

be that there is a component in the enzyme 11 region of the

 

lhO
gel filtration profile which is separable from enzyme 11
by DEAE chromatography, and which is required for a system
of unknown identity which utilizes TPNH and is inhibited
by ATP. This.ATP-inhibited TPNH "oxidase" has the same
type of effect on the assay system as DPNH oxidase. As
seen in Figure 3.3, using components purified by gel
filtration, ATP was not required for the PEP system itself.
In the absence of DPN and ATP the PEP system produces
mannitol-l-phosphate which accumulates so that when only
DPN was added to the incubated assay, TPNH was rapidly
produced in the same amount (as extrapolated in Figure 3.3)
as was produced in the presence of DPN and ATP. This
increase was followed by a net oxidation of TPNH which was
inhibited by addition of ATP so that the rate became al-
most identical to that of the complete system. Also, as
shown in Figure 3.3, in the absence of ATP there was in
this particular case some TPNH production which reached a
plateau value presumably due to the establishment of a
steady state level. The ability to catalyze.ATP indepen-
dent activity may be dependent on the age of the enzyme
11 preparation. Immediately after gel filtration, enzyme
II fractions (such as that used for the data in Table 3.2)
gave very little if any activity in the absence of ATP. Af-
ter two weeks or so of storage at 0°C (after the mercapto-
ethanol was oxidized as indicated by a lack of odor) the

enzyme 11 fractions began to give increasing amounts of

 

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ATP independent activity such as that in Figure 3.3 for
which the same enzyme 11 fraction was used (about two
weeks later) as for the data in Table 3.2. The age of
the enzyme I and HPr fractions did not appear to cause
any change in the ATP requirement. As seen in Figure
3.h the enzyme I fraction gave a linear response in
assays containing ATP in which the other two components
were in excess. However the enzyme 11 fraction, al-
though it gave a fairly linear response at levels up to
that at which the other two components were limiting,
had an inhibitory effect when higher levels were used,
due probably to incomplete inhibition by ATP of the TPNH
"oxidase". Thus when assaying for enzyme I and HPr it
was necessary to use not much more enzyme 11 than that
which was necessary to avoid making it the limiting
component. Using suitably aged enzyme 11 fractions it
was possible to use the mannitol assay without ATP for
analyzing elution profiles for enzyme 1 and HPr (as was
done in Figure 3.1) to give results similar to those for
assays in which ATP was used (as in Figure 3.13 and
Figure 11.2). The amounts of components used in the
assays are stated where necessary. However these Values
vary considerably depending on the factors mentioned above.
The optimal amount of any component and the linearity of

the assay for the component determined had to be checked

for each batch of assays.

 

 

 

lhh

Figure 3.11. INHIBITORY EFFECT OF ENZ II Mtl (gf) .
The mannitol assay (Methods) was used with ATP.

(A) The assay contained 0.01 ml Enz I Mtl (gf IX)

and 0.02 ml HPr Mtl (gf 1X). Enz II Mtl (gf 1X) suas
varied. (B) The assay contained 0.005 ml Enz II 1Mt1
(gf 1x) and 0.02 ml HPr Mtl (gf 1x). Enz 1 Mtl ( gf
1X) was varied. The same batches of enzymes weI‘e
used in both cases. Doubling the HPr did not ezf17€Ct

rates in A or B at the 0.005 and 0.02 ml points.

RATE

RATE

1115

 

 

 

 

 

 

 

 

 

 

Figure 3.“.

5a A

m

g 0 008

E

E

E c

m

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S
0 .005 .01 .02

Enz II mtl (gf 1X) (ml/assay)

5’ B I I

m

m

a.

} 0.008 —— _—

E

\

a: 0.001-P -— ‘1

m

.3

o

‘31

v 0 I I J
0 0.005 0.01 0.02

Enz I mtl (gf 1x)

(ml/assay)

i ... "
“fl“b’

 

 

1&6

Preliminary results suggested that the ATP inhibited
TPNH "oxidase" involves the participation of DPN. Crude
extracts in an assay containing only buffer, 0.0066 M
MgClz, and TPNH had an apparent TPNH oxidase activity
of about 5% that of the DPNH oxidase activity. Addition
of DPN increased TPNH oxidation about 10-fold. It was
also found that an assay for the mannose PEP system
(Methods, Section 3.h.6) which did not contain DPN also
did not have a requirement for ATP. In preliminary work
on the mannose PEP system,an analysis of a Sephadex G200
elution profile of a crude extract of mannitol-grown cells
for enzyme II activity using Enz I F (gf) and a heat
treated, dialyzed (by repeated ultrafiltration)fraction
of HPr F (gf) gave a typical enzyme 11 peak at the
exclusion limit of the profile. This mannose enzyme 11
fraction gave an identical rate when assayed with Enz I
Mtl (gf) and HPr Mtl (gf) fractions. The assay was not
affected by addition of ATP and had a linear time course
and a linear response to enzyme II concentration. Like
the mannitol assay, the mannose assay gave activity in
the presence of only.ATP when crude extracts were tested
but not for gel-filtered components. using only crude
extracts both of the continuous spectrophotometric
assays, at least for quantitative results, were completely
useless. With crude extracts the mannitol and mannose

assays gave plots of rate yg enzyme concentration which

1117
'were nonlinear and variable in shape. This was probably
because it is not possible to use enough extract to
saturate the system with HPr and because of various
possible reactions causing loss of products. It
appears that it is best to ignore continuous spectro-
photometric assays of crude extracts even as a quali-

tative assessment.

3.2.h. PROPERTIES OF THE FRUCTOSE PEP SYSTEM IN CRUDE
EXTRACTS. The FlPK end-point assay for FlP described

in Methods, Section 3.5.3, was used for assays of the
enzyme 11 activity for fructose. Using crude extracts
of fructose-grown cells FlP accumulation was linear
with respect to time for up to at least 10 minutes and
perhaps 20 minutes (Figure 3.19). The variation in rate
with respect to the volume of crude extract of fructose-
grown cells was nonlinear (Figure 3.5). The initial
portion of the curve is proportional to the square of
the enzyme concentration. This is expected since HPr

is probably not at a saturating level for enzyme I at
low crude extract concentrations. Addition of Enz I

Mtl (gf) and HPr Mtl (gf) gave an improvement in
linearity although there was still a small nonlinear
region at low concentrations of crude extract. This
may be due to the dissociation of enzyme 11 for fruc-
tose described below, Crude extracts of mannitol-

grown cells gave a more linear response (Figure 3.5).

 

1118 I

Figure 3.5. FIP FORMATION y§_CRUDE EXTRACT CONCISNTRATION.
The fructose assay (Methods) was used with 0.2 NI fruc-
tose. The volume of the incubation mixture for 'tlie

assay was 1.0 m1. Enzyme components: (1) crmie

extract of fructose—grown cells (0); (2) crude <e><tract

of fructose-grown cells plus 0.175 pmole/’min ozf Enz I
Mtl (gf) and 0.113 pmole/mln of HPr Mtl (gf) (1:1) 3 (3)

crude extract of mannitol—grown cells (A).

Figure 3o5o

lh9

 

0015'“-

0.10

0.05

BATE (uncles FIP/min/assay)

 

 

 

CRUDE EXTRACT
(mg protein/assay)

 

150

In assaying crude extracts it was necessary to keep the
protein concentration in the incubation mixture at about
7 mg/ml or higher in order to ensure that the assay was
limited by enzyme 11, the component of interest in these
experiments. Enzyme II limitation of assays of crude
extracts was checked by obtaining a response to added
gel-filtered enzyme 11 when available, or else by
increasing the concentration of extract to obtain a
proportional increase in activity, indicating that the
assays were not in the nonlinear region of the enzyme
concentration curve.

Extracts of both fructose-and mannitol-grown cells
produced primarily FlP from fructose and PEP. In both
cases only traces of F6P were detectable and G6P or
FDP amounted to about 5% or less of the FlP level. No
activity was observed in the absence of fructose. In
the absence of PEP, accumulation of FlP amounted to up
to about 30% of the level obtained in the presence of
PEP if extracts were used immediately after sonication.
Extracts used two to six hours after sonication (which
is the case for-the data presented here) gave negligible
activity (less than 5%) in the absence of PEP. The PEP-
independent activity is probably due to endogenous for-
mation of PEP. In one experiment using a fresh crude
extract of glucose-grown cells the saturation curve

for fructose in the presence of PEP was identical to

151
that in the absence of PEP except that the rate was about
three times higher.

The fructose saturation curves of crude extracts
grown on various substrates (Figures 3.6, 3.7 and 3.8;
Table 3.h) indicate that there is a constitutive FlP-
forming PEP system with a high Km varying from 0.02 to
0.13 M. The variation was probably due in part to a
lack of enough points in some of the determinations. The
best value for mannitol-grown cells is 0.06 M'and for
glucose-grown cells, 0.02 M (Figure 3.9). The widest
variation in specific activities at 0.2 M fructose was
about two-fold for different batches of cells grown on
the same substrate. Upon induction of cells with fruc-
tose the Km for fructose fell to less than 0.001 M'as
indicated by the typical saturation curves in Figure 3.6.
The atypical saturation curve shown in Figure 3.10 appears
to be composed of two components as derived in Figure 3.11.
A and 3.11.B with the method used by Egan and Morse (89)
for a two component saturation curve for o-nitrophenyl-
galactoside permeation in Staphlococcus aureus, which
°coincidenta11y is probably the Ag_glyg saturation curve
of the lactose PEP system in this organism (96). The
curve in Figure 3.10 is atypical in that it gives the
lowest ratio obtained (see Table 3.h) of activity at
0.002 M fructose to that at 0.2 M fructose. The points
in this curve also appear to be atypically accurate.

The fact that extrapolation of only two points in Figure

152

Figure 3.6. FRUCTOSE SATURATION CURVES OF CRUDE
EXTRACTS OF FRUCTOSEdGROWN CELLS. The fructose

assay as described in Methods was used.

RATE (umoles FIP/min/mg protein)

Figure 3.6.
l V
0.010— _
0.005—' __
o I I I
O 0.02 0.1 0.2

153

 

 

 

 

FRUCTOSE CONCENTRATION

(M)

15h

Figure 3.7. FRUCTOSE SATURATION CURVES FOR CRUDE
EXTRACTS OF MANNITOL-GROWN CELLS. The fructose assay

as described in Methods was used.

Figure 3.7.

155

 

0.015

0.010

(umole s FIP/min/mg protein)

RATE

0.005

 

 

 

0.1

FRUCTOSE CONCENTRATION (M)

 

156

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158

Figure 3.9. LINEWEAVER-BURK PLOTS FOR FRUCTOSE
SATURATION OF CRUDE EXTRACTS OF‘MANNITOL- AND
GLUCOSE-GROWN CELLS. Data are from Figures 3.7
and 3.8. i

Figure 3.9.

<1»

#00

200

159

 

 

  
    
  

glucose-grown

\

\ Manni‘tal-grown
Km ==0.06 M

 

 

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161

Figure 3.10.

:3 2394222828 28822.2

 

 

 

 

Ho.o

No.0

 

 

(uteqoxd Sm/utm/cug 891011111) 33373

 

162

Figure 3.11. LINEWEAVER-BURK PLOTS FOR ATYPICAL FRUC-
TOSE SATURATION CURVE. The points in A are derived from
the experimental points in Figure 3.10. Extrapolation
in A (dotted line) gives the Lineweaver-Burk plot for
the low Km component, the reciprocal values of which
are given in Figure 3.10 as curve.A. Subtracting the
values for curve.A in Figure 3.10 from the experimental
points in Figure 3.10 gives curve B in Figure 3.10,

the reciprocal values of which are given in this figure

as B, the Lineweaver-Burk plot for the high Km component.

 

Figure 3.11.

 

1

Km = 0.00002 M

 

 

 

 

 

 

l
v
o l
o 500 100
.__1_
S]
B l |
/o’
800 — //
/
o //
i ,/ Km 0.05 M
V ///
400 — ’0/
/
O/
O/
o l
o 50 100
.1...
[s]

 

 

16h

Table 3.h. SUMMARY OF FRUCTOSE PEP SYSTEM ACTIVITIES IN
CRUDE EXTRACTS. The fructose assay as described in
thhods was used. Approximate Rulindicates that
reliable assayS‘were obtained only at 0.02 and 0.2 M
fructose. Other 0.002 u/0.2 M ratios obtained for
fructose-grown extracts were 0.63, 0.70, 0.76, 0.77,

all at about 0.01 pmole FlP/min/mg protein at 0.2 M

 

 

fructose.
Specific
Growth .Activity Rate at
Substrate pmoles/min/ 9.002 M fructose Km (M)
mg protein at Rate at

0.2 M fructose 0.2 M fructose
”

 

Fructose 0.005? I 0.98 * -'
" 0.0072 “' -‘
" 0.010 0.75 less than 0.001
" 0.0085 0.79 less than 0.001
" 0.01u 0.59 0.00025 + 0.013
Mannitol 0.010 — _—
" 0.0098 0.00 0.16 (approx.)
" 0.019 0.16 0.0h (approx.)'
" 0.0060 -— 0.0h (approx.)

" 0.0083 0.03 0.06

 

165
Table 3.h. (continued)

 

 

Specific
Growth Activity Rate at
Substrate pmoles/min/ 0.002 M fructose Km (M)

mg protein at Rate at
0.2 M fructose 0.2 M fructose

 

 

 

 

 

 

 

Glucose 0.0069 —— .__

" 0.0060 0.16 0.07 (approx.)

" 0.00h5 0.11 0.02
Glycerol (0.00h2 ——’ -——

" 0.003u 0.25 - 0.025 (approx.)
Nutrient
Broth 0.0010 -—- -—-

" 0.00u8 0.21 0.08 (approx.)
Sorbitol 0.0026 -—- ._—
Mannose 0.0031 .— __
L-arabinose 0.0030 -—- --

 

*
An unusually high concentration of crude extract was

used (15 mg protein per ml of incubation mixture

instead of about 7 mg protein per ml).

166

3.11.A results in the unique resolution of the two compo-
nents (as indicated by a linear plot in Figure.3.ll.B)
must be attributed to chance. Nevertheless the analysis
indicates that the Km of the low'Rm component may be as
low as,or lower than,2 x IO'h M and that the high Km
component has a Km which is close to that of a component
'which appears during purification, as described below.
What determines the extent to which a high Km component
appears in the fructose saturation curves of crude
extracts of fructose-grown cells is not known; it may
involve the concentration of extract in the assay or
other factors (see Discussion, Section 3.3.2). However,
considering the typical saturation curves it appears that
there is not enough of the high Km activity component
present in these curves to account for the constitutive
high Km system. This suggests that the high Km system

is either repressed or inhibited during growth on fruc-
tose,or else that it is converted to the low'Km system.
When extracts of fructose and glucose-grown cells were
mixed it was found that the activities were not additive,
the results being consistent with a partial conversion

of some sort of the high Km system in glucose-grown cells

to a low Km system (Figure 3.12).

167

Figure 3.12. NON-ADDITIVITY OF ACTIVITY OF CRUDE
BXTRACTS OF FRUCTOSE-AND GLUCOSE-GROWN CELLS. The
fructose assay (Methods) was used, with 0.2 m1 of
an extract of fructose-grown cells (0), 0.2 ml of
an extract of glucose-grown-cells (Cl), and 0.1 ml
of both (A), per ml of incubation mixture. If

the activities had been additive the points for the

mixture would have fallen on the dotted line.

RATE (umoles FIP/min/mg protein)

168

Figure 3.12.

 

| 1

 

 

I | I

 

 

0 0.02 0.1 0.2

FRUCTOSE CONCENTRATION (M)

169

3.2.5. COMPARATIVE PURIFICATION OF THE MANNITOL AND
FRUCTOSE PEP SYSTEMS -— APPARENT MJDIFICATION OF THE
.AFFINITY OF ENZYME II FOR FRUCTOSE. Using the mannitol
assay it was possible to purify the components of the
fructose PEP system. Gel filtration separations were
made for extracts of mannitol-grown and fructose-grown
cells (Figure 3.13). Analysis of the elution profile
of the extract of mannitol-grown cells gave peaks for
enzyme II NR1, enzyme I and HPr. Using the Enz II Mtl
(gf) fraction,the elution profile of the extract of
fructose-grown cells was assayed for enzyme 1 and HPr
giving similar peaks. Analysis of this elution profile
for enzyme 11 Mtl revealed no activity at 0.0033 M
mannitol. Analysis of the two elution profiles for
enzyme II using 0.2 M fructose, Enz I Mtl (gf) and

HPr Mtl (gf) gave peaks in the enzyme 11 region of
both elution profiles.

The affinity of Enz II F (gf) was found to be
determined by the source of the enzyme 1 and HPr frac-
tions (Figures 3'1h: 3.15, and 3.16 and Table 3.5).

With enzyme 1 and HPr fractions derived from the extract
of fructose-grown cells, Enz II F (gf) gave a saturation
curve similar to that found using crude extracts of fruc-
tose-grown cells. However using fractions derived from
nmnnitol-grown cells the Km for fructose was about 0.07

lehich is in the constitutive range. For Enz II Mtl

 

 

 

 

 

170

Figure 3.13. ANALYSIS OF GEL FILTRATION ELUTION PROFILES
OF FRUCTOSE-AND MANNITOL-GROWN CELLS. A 70 m1 sample of
crude extract of cells grown on mannitol, (A), or
fructose, (B) was chromatographed on the same 700 ml
bed of Sephadex G100 (with mercaptoethanol). Prelimi-
nary recombining of fractions established the approxi-
mate location of the PEP system components. Assay types
and components (see text for summary): (1) Fructose
assay for enzyme II (0) - 0.2 ml total volume, 0.02 ml
Enz I Mtl (gf 10X), 0.02 ml HPr Mtl (gf 10X), 0.2 M
fructose. (2) Mannitol assay for enzyme 11 (O) - 0.005
ml Enz I Mtl (gf 10X), 0.01 ml HPr Mtl (gf 10X). (3)
Mannitol assay for enzyme I (A) — 0.005 ml Enz II Mtl
(gf 1X), 0.01 ml HPr Mtl (gf 10X). (h) Mannitol assay
for HPr (ED - 0.005 ml Enz II Mtl (gf 1X), 0.005 ml
Enz I Mtl (gf 10X). (5) Fructose assay for the Km factor
(dotted line) ~ 0.22 ml final volume, 0.0u ml Enz II F
(gf 1X), 0.02 ml Enz I Mtl (gf 10X), 0.02 ml HPr NR1
(gf 10x), 0.1 m1 of Km factor fraction. With this assay
the ratio of activity at 0.002 M fructose to that at 0.2
M fructose for each fraction assayed was taken as the Km
factor index value.

All components above are from these two elution pro-
files. The volume ranges used for the final component
pools were: Enz II Mtl (gf 1X) 210-2h0 ml from A; Enz I

Mtl (gf 10x)380-u90 m1 from A; HPr Mtl (gf 10x) 510—670

 

171

Figure 3.13.

mm 30am “)1

m.) e ,

RI. f.

4225 hoooom am

5.1.
H sauna

 

 

 

 

 

ImmOEODmm . m

_

 

32 HH cannon

zsomw

 

_

 

\ an:
H manna HH manna
.2 HH manna
4
zzomleoaHZ—fiz .4

_ _

   
 

 

«.0

N5

m.o

 

(Infant/s atomn) m

172

Figure 3.13. (continued).

ml from.A; Enz I Mtl (gf 10X) 380-h90 ml from.A; HPr
Mtl (gf 10X) 510-670 ml from.A; Enz II F (gf 1X) 210-
2h0 mi from B; Enz I F (gf 10X) 390-h90 ml from B;
HPr F (gf 10X) 510-690 ml from B. Km factor samples
for other experiments (as noted) were taken from
unpooled portions of tubes in the REG-500 ml region
of B.

Table 3.5.

173

SUMMARY OF KINETIC PARAMETERS OF ENZYME II

FOR FRUCTOSE. Data are from Figures 3.15 and 3.16.

 

PEP System Components Vmax for
umoles/min .Fructose (M)
Enzyme II Enzyme I HPr ml Enz II
Fructose Fructose Fructose 0.39 0.001 or less
Fructose Mannitol Mannitol 0.20 0.070
Mannitol Fructose Fructose 0.h2 0.06M
annitol Mannitol Mannitol 0.37 0.06h

 

 

v ., ,p. arr—Via ‘

M} w" '

17h

Figure 3.114.. FRUCTOSE SATURATION CURVES USING GEL-

FILTERED COMPONENTS. The same batches of componentS'

as described in Figure 3.13 were used. Components for

fructose assays for enzyme 11 (0.5 ml final volume):

(A) 0.1 m1 Enz II F (gf 1X), 0.1 ml Enz I F (gf 10X),
0.05 ml HPr F (gf 10X);

(B) 0.1 m1 Enz II F (gf 1X), 0.1 ml Enz I Mtl (gf 10X),
0.05 ml HPr Mtl (gf 10x):

(C) 0.1 ml Enz II Mtl (gf IX), 0.1 m1 Enz I F (gf 10X),
0.05 ml HPr F (gf 10x);

(D) 0.1 m1 Enz II Mtl (gf 1X), 0.1 ml Enz I Mtl (gf 10X),
0.05 ml HPr Mtl (gf 10X);

(E) 0.1 ml Enz II F (gf 1X), 0.2 m1 Enz I F (gf 10X),
0.1 m1 HPr F (gf 10x);

(F) 0.1 ml Enz II F (gf 10X), 0.2 ml Enz I Mtl (gf 10X),
0.1 m1 HPr Mtl (gf 10X).

175

Figure 3.14.

BATE (umoles FIP/min/ml Enz II)

 

  
  
 
  
 

l l
(E) Enz II F
Enz I F (2x)"““-
HPr F (2x)

\\
(A) Enz II F
Enz I F, HPr F

   
   
 
   

 

031'—

(D) Enz II Mtl ’
Enz I Mtl
HPr Mtl

(0;\Enz II Mtl
Enz I F
HPr F

(F) Enz II F
Enz I Mtl (2x)
HPr Mtl (2x)

 
     

l

(B) Enz II F
Enz I F
HPr F

 

o l l l

 

 

0 0.02 0.1 0.2
FRUCTOSE CONCENTRATION (M)

 

176

42$“ 0.3m; Ea; 0.2m mama

E \
.mZOHH/SIZHmEOU x IOHE mOm ”H.013 xgmlmmK/QMSMZHA .mHJ. mpsmwm

177

Figure 3.15.

I'_I
U)
1.!

.5

00 om 02 00 00

 

000.0 n 22 2 222
.2 H N02 .202 HH Nam

200.0 n 22
202 222;
.202 H N22 .202 22 New

020.0 n 22
2220 202 222
.320 H022 H Nnm .LHH N05. 1

ll

 

                

 

0.“

ON

178

Figure 3.16. LINE1.’.’EAVER-BURK PLOT FOR LOW Pg,” COMBINA-

TION. Data are from Figure 3.1M.

179

Figure 3.16.

 

 
 
  

Enz II F,
Enz I F (1x)
HPr F (1x)

 

 

 

$1 Km 2 less than 0.001 M
V
2.02—-
o l I J I l
0 100 200 300 400 500

1

w

[s]

180

(gf) the Km values were in the constitutive range and were
the same for enzyme I and HPr from both elution profiles.
The Km of the Enz II Mtl (gf) fraction for mannitol using
the mannitol assay was about 5 x 10-5 M (:50%) using gel—
filtered components from mannitolngrown cells. No detect-
able change was observed using components from fructose—
grown cells. However assays at such low levels of sub—
strate were rather inaccurate so that a small change
would not have been detected. Assays of a fraction from
the center of the enzyme 11 region of the elution profile
of the extract of fructose-grown cells (Figure 3.13.A)

at 0.066 It mannitol gave no activity indicating an
absence of a high Knlmannitol system.

Further testing of the enzyme I and HPr fractions by
mixing them in the four possible combinations [Enz I F
(gf) plus HPr Mtl (gf), etc.] indicated that the factor
responsible for the apparent shift in Km (the "Km
factm”Vlwas located somewhat behind enzyme I in the eluw
tion profile of the extract of fructose—grown cells.
Testing the fractions in this region with an assay
designed to detect the Km shift gave the peak indicated
in Figure 3.13.B. For this assay Enz II F (gf) was
coupled with Enz I Mtl (gf) and HPr Mtl (gf) to give a
high Km, enzyme II limited assay. Addition of the Km
factor caused a shift toward the low Km type of system.

Assuming that a simple Michaelis-Menton saturation

181

curve is involved, the Km factor index plotted in Figure
3.13'which is the ratio of activity at 0.002 M fructose
to that at 0.2 M fructose, is‘a nonlinear function of

the Km which approaches unity as the Km approaches zero.
Hawever the detailed kinetics of the enzyme 11 fructose
system have not been examined so that it is quite
possible that a compound Michealis-Menton system, con-
sisting of more than one species of enzyme II, may be
involved in the presence of the Km factor, particularly
when it is not at a saturating level. Thus the Km factor
index is used only as an emperical indicator of the
presence of the Km factor. The reaponse of the Km factor
assay to increasing enzyme was linear as seen in Figure
3.17. Note that the net increase in activity at 0.002

M was greater than that at 0.2 M. This is consistent
‘with at least a partial conversion of the high Km system
to the low'Km system rather than simple addition of a

low Km system to the high Km system.

3.2.6. REQUIREMENTS OF THE FRUCTOSE PEP SYSTEMS. Using
gel-filtered components,G6P and F6P were not detectable
in incubation mixtures using either Enz II F (gf) or

Enz II Mtl (gf) fractions.. FDP occurred at about 5% or
less of the PIP level. The requirements for different
component combinations are given in Table 3.6. No loss
of added F1? was observed for a 10 minute incubation in

the absence of PEP. For the assay using Enz II Mtl (gf),

 

 

182

Figure 3.17. EFFECT OF Km FACTOR FRACTION. The
fructose assay contained in a final volume of

0.95 ml; 0.1 m1 Enz II F (gf IX), 0.05 ml Enz I
Mtl (gf 10X), 0.1 ml HPr Mtl (gf 10X), 0.002 M

or 0.2 M fructose as indicated and the indicated
volume of Km factor fraction. The components were

those described in Figure 3.13 except that a

 

different Enz II F (gf 1X) fraction from the same

profile was used.

183

Figure 3.17.

 

0.3 —— __

 

 

 

 

E:
H
H
r"! -" 100 8 °
.5 .
\E V
H 0.002 M >4
8 B
\ z
81 H
[In
m ‘5
Q) 9
rr; 3
a In.
3 x
E3 —- 0.5

0 I I O

O 0.25 0.50

Km FACTOR FRACTION (ml/assay)

18h
Tabl3 3.6. REQUIREMENTS FOR THE FRUCTOSE ASSAY USING GEL-
FILTERED COMPONENTS. Assays were as in Figure 3.13 using
the same components except that enzyme II fractions were
from somewhat different regions of the elution profiles

in Figure 3.13. Assays contained 0.2 M fructose.

 

Assay Relative
Components Assay Type Rate
l.* Enz II F complete 100%
Enz I Mtl " minus PEP O
HPr Mtl " minus fructose O
" minus Enz II F 6
" minus HPr Mtl O
" minus Enz I Mtl O

" minus Enz I Mtl,minus

HP? Mtl 0
minus Enz II F;plus

Km factor (0.1

ml/O.S ml assay) 0
" minus PET,plus ATP

(0.002 ML'plus‘
Mg012 (0.00Li M) m

Complete assays, minus fructose and PEP, plus FlP
(0.000hhqnonfinal), after incubation for zero and
10 minutes contained 0.161 and 0.168 pmoles FIR/0.5

m1 assay respectively.

 

 

185

 

 

Table 3.6. (continued)
Assay Relative
Components Assay Type Rate
2. Enz II Mtl complete 100%
Enz I Mtl " minus PEP O
HPr Mtl " minus fructose 0
" minus Enz II Mtl 6
" minus HPr Mtl l9
" minus Enz I Mtl 15
" minus Enz I Mtl,
minus HPr Mtl l
3.* Enz II F complete 100%
Enz I F " minus PEP 0
HPr F " minus fructose 0
" minus Enz II F 0

Complete assays, minus fructose and PEP, plus FlP

(0.000h M, nominal), after incubation for zero and

10 minutes contained 0.185 and 0.180 pmole F1P/O.S

ml assay.

 

 

h. Enz II Mtl complete

Enz I F " minus

HPr F

PEP

100%

 

186

Enz I Mtl (gf) and HPr Mtl (gf) some activity was observed
in the absence of either Enz I Mtl (gf) or HPr Mtl (gf).
This was because there was cross contamination of these
two fractions due to the overlap of enzyme 1 and HPr in
the regions of the elution profile which were pooled

for these particular fractions. Use of narrower fractions
or further purification of fractions eliminates this
activity. However it is not known why the same results
were not obtained using these same fractions with Enz II

F (gf) (Table 3.6)° Assays employing gel—filtered com=
ponents gave linear responses to increasing amounts of

Enz II F (gf) and Enz II Mtl (gf) (Figure 3.18). As
determined with the mannitol assay, the enzyme I~HPr
activity used in these assays was 5 to 20 times that of
the enzyme II used. As discussed below, however, it was
found that the most important criteria for an enzyme II
assay is a lack of a substantial increase in rate when
Enz 1 and HPr are doubled, as confirmed for the data in
Figure 3.1M.

The pH of assay mixtures containing gel-filtered
components was tested for each new batch of components
and was found to vary from 7.52 to 7.55. Assays of
crude extracts varied in pH from 7.h to 7.55 but there
was no coincidence between pH values and affinity for
fructose. The pH did not change significantly during

the incubation or even after heating to remove protein.

 

 

 

Figure 3.18. RATE OF FlP FORMATION Y§ VOLUME OF

ENZYME II FRACTION. Fructose assays were like

those in Figure 3.1M at 0.2 M fructose. Assays:

(I) For Enz II Mtl (gf IX) using Enz I Mtl (gf 10X)
and HPr Mtl (gf 10X) (0); (2) for Enz II F (gf 1X)
using Enz I Mtl (gf 10X) and HPr Mtl (gf 10X) (ED;
(3) for Enz II F (gf 1X) using Enz I F (gf 10X) and~
HPr F (gf 10X) (A). For assays (l) and (2) the enzyme
I and HPr fractions were those used in Figure 3.13
and enzyme II was from slightly different fractions
from the same elution profiles. For assay (3) all
components were from different batches than those

used in Figure 3.13.

188

Figure 3.18°

RATE (umoles FIP/min/assay)

.08

.06

.04

.02

 

 

 

 

\ (2)

l

 

 

001
Enz II F or Mtl (ml/assay)

_ .__... ._1

189

variation of the PEP concentration by 2 and 0.5 fold
also gave no significant changes in rates using crude
extracts of mannitol-grown cells. The time course of
the reaction using gel-filtered components was linear
up to at least 10 minutes (Figure 3.19).

It can be seen in Figure 3.13 that the Km factor
does not coincide exactly with enzyme I. A more
complete analysis (Figure n.2, Section h.2.2) gave
similar results. That the Km factor is not an enzyme
I was also indicated by the fact that enzyme 1 F - Km
factor fractions if stored for several weeks lost al-
most all enzyme I activity but retained full Km factor
activity. Purification of enzyme I F — Km factor frac-
tions from gel filtration on DEAE cellulose (Figure
3.20) resulted in loss of the Km factor activity and
recovery of the enzyme Ilactivity as determined by the
mannitol assay. In this experiment the loss of the
factor may be due to dilution to such an extent that it
was not detectable. However had it undergone the same
dilution as did enzyme I it should have been at a high
enough level to detecto There was some indication in
this experiment of either a shoulder or a skewed region
.on the forward side of the enzyme I peak.

There is some preliminary evidence suggesting that
multiple enzyme I — HPr systems exist. One case is the

Observation of Tanaka and Lin (87) that as determined by

 

190

Figure 3.19. TIME COURSE OF FIP PRODUCTION. The
fructose assay (Methods) was used with (l) a crude
extract of fructoseagrown cells (0.2 ml per 1.0 ml
assay) (D) and (2) Enz Ii F (gf) (at a rate—limiting

concentration, 0.0h ml per 0.587 ml assay), Enz I F

(gf) and HPr F (gf) (O).

 

 

191

Figure 3019

 

 

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5
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192

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Figure 3.20.

193

 

 

 

 

 

 

(ovum/Imam Seton“) I mums
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1911

an assay utilizing enzyme 11 for mannitol, loss of enzyme

I in A. aerogenes (strain 1033) causes loss of growth on

 

glycerol whereas loss of HPr does not. Interpretation
of this is difficult since neither of the two pathways
which had previously been established for glycerol
metabolism in this strain of A, aerogenes involved an
unknown phosphorylation which might now be attributed
to a PEP system (97). Fructose metabolism is impaired
in both of these pleiotropic mutants although there is
somewhat greater growth in the HPr-negative mutant as
indicated by patch testing on agar. This would indicate
that at least in this strain the same types of enzyme I
and HPr that support the mannitol system probably also
support the fructose system.

In attempting to eliminate the possibility that a
multiple enzyme I - HPr system could be responsible for
the Km shift the major consideration is that such a

system would definitely require the presence of two
species of HPr, one for the high Km enzyme II and one
for the low'Km enzyme II. From Sephadex 6200 chromato-
graphy it appears that the molecular weight of the Km
factor is too high for it to be a typical HPr. In

Table 3.6 it can be seen that the Enz II F (gf), Enz I

F (gf), HPr F (gf) assay has a requirement for the HPr

F component.' Thus the Km factor which is present in the
Enz I F (gf) fraction does not furnish the HPr for the

195

reaction.

Suppose however that the HPr F (gf) fraction actually
contains a special enzyme I for the fructose system and
that there is in the Enz I F (gf) fraction 3 Special HPr
for the lowKm fructose system (i.e. the Km factor). In
this case the special enzyme I would have to be in the
HPr Mtl (gf) fraction since low Km systems can be obtained,
in the presence of the Km factor, which are dependent on
this fraction. However since the HPr Mtl (gf) fraction
retains activity after heat treatment when used in the
enzyme II F - Km factor system (see Table 3.9 discussed
below) it is probably functioning as an HPr rather than
as an enzyme I supply since an enzyme I would not be
expected to be heat stable. The Km factor is definitely
not an enzyme II or any other type of PEchexose phos-
photransferase as seen by the lack of activity of an
assay lacking enzyme II (Table 3.6) and also by the fact
that the enzyme II assay used in Figure 3.13 did not give

a peak in the Km factor region.

3.2.7. APPARENT NONIDENTITY OF HIGH Km ENZYME II SYSTEMS.
annitol inhibits the formation of FlP catalyzed by the
Enz II Mtl (gf) fraction (Table 3.7). The inhibitory
effect was saturated at 0.002 M mannitol and appeared to
involve about 60% of the total high Km activity.

Whether or not the K1 is the same as the very low Km for

mannitol of the mannitolwl-phosphate forming activity

 

 

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197

catalyzed by this fraction could not be tested, since
even at 0.002 M substrate the assay for the formation

of F1? requires that about 25% of the sugar be converted
to sugar phosphate in order to obtain accurate assays for
FlP. The Enz II F (gf) fraction was not inhibited by
mannitol regardless of the source of enzyme I and HPr.
The possibility that the inhibition might be due to
competition for phospho-HPr by the mannitol-l-phosphate
forming system (even though there was about a lO-fold
excess of phospho-HPr forming activity in these assays)
was eliminated by finding the same inhibition at a lower
concentration of Enz II Mtl (gf). In Table 3.8 are

given some preliminary results of a survey of patterns
of inhibition of the high Km activity using crude
extracts. In this case phOSpho-HPr limitation may be
responsible for some of the effects. Mannitol inhibited
the activity only for extracts of cells grown on mannitol,
which were also inhibited by sorbitol and mannose. annose
appeared to inhibit somewhat in extracts of cells grown
on several substrates. DEAE cellulose chromatography of
Enz II F (gf) as compared to that for Enz II Mtl (gf)
(Figure 3.2) also suggests that the two high Km systems
are due to different proteins. The small peak of activ-
ity in the enzyme II Mtl profile could possibly be that
component in the Enz II Mtl (gf) fraction which is not

inhibited by mannitol rather than an artifact due to a

198

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199
discontinuity in the elution.

Thus it appears that there are possibly three types
of high Km FlP forming enzyme II systems. (i) The manni-
tol inhibited component which is unresponsive to the Km
factor, which forms only FlP and which is probably the
same component that forms mannitol—l-phosphate and is
induced by mannitol. (ii) The Km factor responsive
component in the Enz II F (gf) fraction which also forms}.
only FlP. And (iii), the constitutive high Km system
in cells grown on glucose, glycerol, sucrose, nutrient
broth and other substrates and which has not been

purified.

3.2.8. ACTIVATION BY'THE Km FACTOR AND PURIFICATION OF
ENZ I Mtl (9f). The Km factor consistently caused an
activation of the activity of the Enz II F (gf) fraction
at 0.2 M fructose. Purification of the Enz I Mtl (gf)
fraction increased the degree of activation. For the
data in Table 3.9 an Enz I Mtl (gf) fraction was purified
with a calcium phosphate gel treatment as described in
Methods. The purification resulted in a good recovery
(83%) of enzyme I activity and resulted in a five-fold
purification above the gel filtration level as determined
with the mannitol assay. When this preparation was used
(with heat treated HPr) in tests of the activating effect,
the activation at 0.2 M fructose was about three times

greater than that normally observed with gelufiltered

 

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20?
components. This was because there was about a three-fold
loss of activity using the purified enzyme I fraction in
the absence of the Km factor. The activity of Enz II F
(gf) in the presence of the Km factor was not appreciably
changed by the purification. The Enz II Mtl (gf) fraction
‘with the purified enzyme I fraction still gave no response
to the factor at 0.2 M fructose and although its activity
“ms not checked with the Enz I Mtl (gf) fraction used in
these experiments it was about the same as that observed
using other batches of enzyme I Mtl (gf). When the
purified enzyme I NR1 fraction was tested for ability
to saturate an assay for Enz II F (gf) in the absence
of Km factor (Figure 3.21) it was impossible to saturate
the system, there being a linear response to increments
of the purified enzyme I Mtl fraction even at enzyme I
levels which, as determined with the mannitol assay, were
far in excess of the activity observed. Using the same,
apparently enzyme I limited system, it was found (Figure
3.22) that the reSponse to increasing Enz II F (gf) was
linear both with and without the Km factor. (The Km
factor index values obtained in this experiment approach
100%, the highest values observed using purified compo-
nents). Considering the two equations describing the
relationship between enzyme II, enzyme I and HPr (Intro-
duction, Section 3.1) it is apparent that it is possible

to have a single assay system which is linear with

my)..-

203

Figure 3.21. RATE y§ ANDUNT OF PURIFIED BN2 I Mtl.
The fructose assay was as described in Table 3.9
using Enz II F (gf) in the absence of Km factor with
increasing amounts of calcium phosphate gel purified
Enz I Mtl. The dotted line indicates the plot if a

normal enzyme I limited assay had been obtained.

20h

 

 

 

ENZ I (Gel) (umoles/min/assay)

Figure 3.21.
0.02 __ /
:5: /
3 /
Q
E /
a /
“‘4 /
§ 0.01 ... /
i—i /
g /
3 /
/
/
o / 1 I
0 0.1 0.2

 

 

H...

205

Figure 3.22. RATE vs AMOUNT OF ENZ II F USING PURIFIED
ENZ I Mtl. Fructose assays were as described in Table
3.9 using increasing amounts of Enz II F (gf 1X); with
and without Km factor; at 0.002 M and 0.2 M fructose;

and using Enz I Mtl purified with calcium phosphate gel.

 

Figure 3.22.

RATE (umoles FIP/min/assay)

206

 

 

0.2 M Fructose o
+ Km Factor

0.002 M
Fructose +
Km Factor

   
    
 

0.2 M Fructose

- Km Factor

0.002 M Fructose
— Km Factor

 

0.01 0.02

ENZ II F (ml/assay)

 

207

respect to both enzyme I and HPr, the linearity of HPr
being due to the first‘order portion of the HPr satura-
tion curve for enzyme I. However it is not possible to
have a single system which is linear with respect to both
enzyme II and enzyme I (or HPr). Therefore in assays
containing the purified enzyme I the linear response

of either the Enz II F (gf) fraction in the absence

of the Km factor or else that of the purified enzyme

I fraction must be of an abnormal character. An explana-
tion is that the enzyme II component in the Enz II F
(gf) fraction is actually completely inactive in the
presence of completely purified enzyme I and HPr but
that there is in the Enz I Mtl (gf) fraction a component
'which activates this enzyme II component via interaction
similar to that shown by the Km factor, but which unlike
the Km factor in fructose-grown cells results in a high
Km FlP forming system similar but not necessarily
identical to that in mannitol-grown cells. (This compo-
nent might be thought of as a "high Km factor".) A
substantial loss of this component during calcium phos-
phate gel purification would cause the observed loss of
Enz II F (gf) activity in the absence of the Km factor.
Furthermore the purified enzyme I fraction would give a
linear response regardless of the amount of enzyme I
present since it is also supplying a limiting component

necessary for enzyme 11 activity. The Enz II F (gf)

208

component, provided that the interaction with the "high
Km factor" were reversible, would also give a linear
response. If the system were saturated with Km factor,
the purified enzyme I fraction would no longer be a
limiting component and Enz II F (gf) would be more active.
The data are consistent with every aSpect of this line

of reasoning. It is possible that in crude extracts of
cells grown on substrates other than fructose a "high

Km factor", apparent only in the presence of highly

purified enzyme I and heat treated HPr, might be found.

3.3. DISCUSSION

The original purpose of this portion of the inves-
tigation, to find the in EEKE source of FlP for metab-
olism via FlPK, appears to be satisfied by the present
findings. The specifically induced property of the PEP
system for fructose, its high affinity for fructose and
its specificity for production of the l-phosphate are
consistent with an lfl.XlE£.r01€ in fructose metabolism.
Confirmation of the in 3132 involvement ofthe PEP
system and the Km factor in fructose metabolism was ob"
tained in the analysis of a mutant which lacks the Km
factor as described in Section h.2.2. In the present

discussion, we consider the role of the Km factor in

the fructose enzyme II system.

209

3.3.1. POSSIBLE EFFECTS PRODUCING AN ARTIFACTUAL Km
FACTOR. Several possible situations which might create
an apparent Km factor must be considered. The results
indicate that multiple enzyme I-HPr systems of the

most likely types are not reSponsible for the Km factor
effect. However it is found that this interpretation
and any other along the same line depends on the
assumption that the system involved is basically
similar to to that in E, 2211 as characterized by
Kundig, Gosh and Raseman (82). This system is the only
one for which it has been rigorously established that
the three PEP system components are related as
described by the two usual equations for PEP systems
(Introduction, Section 3.1) although there is a pre-
liminary report for the character of HPr in Sallmonella

typhimurium (98). Thus all of the current work in

 

this field relies upon an analogy between the E, coli

system and those in other organisms. For A, aerogenes
this analogy is supported by the finding that the enzyme
II for mannitol is active when supplied'with enzyme I
and HPr purified from E, 2311 (85). However the fact
that the status of enzyme I and HPr is at present in a
rather confused state due to unexpected anomalies found
in the characterization of enzyme I and HPr negative
mutants (see reference 83 for review), indicates that

a multiple enzyme I-HPr system of an unusual type

210

cannot be dismissed from consideration.

Is it possible that FlPK, an FlP phosphatase or
other system, by utilizing FlP could cause an apparent
drop in the Kb? In Figure 3.21 are illustrated the
effects of the two basically different types of product

utilizing systems -— namely those in which the Vmax

of the utilization system is higher than that of the

PEP system and those in which it is lower. It can be
seen that the high Vmax system gives no change in
apparent Km and produces a nonlinear time course which
was not observed in the time period used for the assays,
thus eliminating it from consideration. It should be
noted however, that it is quite possible that there is,

potentially,a high Vha utilization system which is

x
avoided under the conditions used in the assays for

FlP formation. If the FlP concentration in the incuba-
tion mixtures for crude extracts had been allowed to
rise to a high enough concentration, ultimately loss

of product due to various known phosphotransferase
reactions (Section 2) and perhaps other unknown re-
actions might have occurred. If this were the case the
assay for FlP formation, since it was linear with
respect to time, would be functioning in that region on
the time axis in Figure 3.23.A between 0 and the point

marked by the arrow, i.e., that region which gives

valid assays. The low'V’max system causes an apparent

“I“. :i-c—w—v'”. "it"

211

Figure 3.23. THEORETICAL EFFECT OF PRODUCT LOSS ON
OBSERVED Km. For both.A and B the same time course
for a PEP system with a real Km factor index value

of 0.3 is assumed, as indicated by the dashed lines
for real FlP production at 0.2 and 0.002 M fructose.
The observed rates were derived by graphically sub-
tracting,at short time intervals from the real rate,
the rate of product loss due to an FlP utilizing
reaction at the FlP concentration at each time inter-
val used. The observed index values, indicating the
effect on the observed Km, are calculated from the
observed time course plots. In both A and B the Km
of the utilization system is 12.5 FlP concentration
units. The vmax of the FlP utilizing system is in A,
2.0 times, and in B, 0.2 times the real rate of the
PEP system at 0.2 M fructose. The arrows indicate the
time points up to which the observed and real results
are approximately the same. Changing the Km of the
FlP utilizing system for FlP has an effect similar to
that caused by changing the scale of the axes for
time and FlP concentration, i.e., there is no change

in the basic shape of the curves.

W

Figure 3.23.

FIP CONCENTRATION
IN.ASSAX

FIP CONCENTRATION
IN'ASSAY

30

N
O

100

 

212

 

 

 

 

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//
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213

increase in Km rather than a decrease making its presence
unlikely. In this case a break in the time course of the
reaction is observed only if the Km for FlP of the
utilization system is high enough, otherwise the time
course could be apparently linear. This type of inter-
ference is ruled out for gel-filtered components by

the stability of FlP in these systems (Table 3.6)
indicating a lack of phosphatase action, the only known
FlP utilizing system which might be present in cells
grown on sugars other than fructose. It also appears
that the low‘V’max type of utilization would not be able
to raise the Km of a system with a high Km factor index
ratio without causing an almost total loss of activity
at 0.2 M fructose using crude extracts of cells grown

on substrates other than fructose.

Is it possible that by exerting a simple activating
effect the Km factor could, without having a true effect
on substrate affinity, create an artifactual decrease in
Km by causing the activity at 0.2 M fructose of a high
Km system to be so high that much of it is not observed
due to a limiting effect of the enzyme I-HPr system?

This is ruled out by the observation that doubling the
enzyme I and HPr concentration did not effect the satura-
tion curve (Figure 3.1h). It is also ruled out by the
linear response of Enz II F (gf) in the presence of the

factor at both 0.2 and 0.002 M fructose as seen in

211.

Figure 3.22.

3.3.2. MODELS FOR ENZYME II ACTIVITIES. Three models
can be proposed for the fructose enzyme II system which,
depending on their complexity,give an increasingly
better fit with the data.

A. Inducible enzyme II base model. The simplest
model is the relationship:

Enzyme 11 base (inducible) + Km factor (inducible)

;===3 Enzyme II base-Km factor complex
The enzyme II base is a large membrane bound enzyme
which is present in the Enz II F (gf) fraction. In the
presence of theKm factor an active enzyme II complex is
formed which has a lowKm for fructose. This model would
be in accord with the data if it were not for several
varieties of high Km enzyme II activities and other
effects._

B. Constitutive enzyme II base model. A somewhat
better fit with the data is obtained if it is proposed
that the enzyme II base is a constitutive enzyme, rather
than a subunit of an inducible enzyme, and that in the
absence of the Km factor it catalyzes FlP formation with
a high Km for fructose, thus explaining the high Km consti-
tutive fructose PEF activity. Interaction with the
inducible Km factor modifies the active site causing an

increase in both the affinity for fructose and the Vmax

215

of the reaction. This modification is similar to the
role of the "specifier protein", colactalbumin, in the
lactose synthetase system (99). In analogy the Km
factor is designated the fructose specifier protein in
the reaction:

Enzyme 11 base (constitutive) + Fructose specifier

(inducible):;==§ Enzyme II base —— Fructose

specifier protein.

C. Common enzyme II base model. A complete fit
with the data is obtained if it is proposed that the
enzyme II base is common to more than one enzyme II
activity as diagrammed in Figure 3-2h- The enzyme II
base is a constitutive component which is completely
inactive by itself and which, by interaction with any
one of several specifier proteins, produces complexes
which catalyze enzyme 11 activities Specific for cer-
tain substrates. This model is proposed essentially
as a mechanism by which the substrate specificity of a
set of enzyme II activities might be determined. The
Km factor is the inducible specifier protein producing.
the low'Km enzyme II complex for fructose. At least
some of the other enzyme 11 complexes, in addition to
their specified reactions, catalyze, as a nonspecific
reaction similar to those catalyzed by other phospho-
transferases, a high Km fructose enzyme II activity --
to a variable extent and with perhaps somewhat variable

affinities for fructose. These side reactions constitute

111.. 1.1.3.

‘ldl‘iwr 0 O
IN . o

216

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217

the constitutive enzyme II activity for fructose. The
Enz Mtl (gf) fraction contains an enzyme II base-
mannitol specifier protein complex which, unlike the
enzyme II complex for fructose, does not dissociate
appreciably under the conditions used so that a "mannitol
Km factor" is not observed and the fructose-induced Km
factor does not interact with the Enz II Mtl (gf) frac-
tion. The otherwise rather peculiar mannitol inhibition
of the high Km activity of the Enz II Mtl (gf) fraction,
is thus an expected consequence of the proposed structure
of enzyme II for mannitol. It may also be suggested
that the low, uninduced, basal levels or appreciable
constitutive levels of specifier proteins which are
responsible for the constitutive fructose enzyme 11
activity are also present in the Enz I Mtl (gf) fraction
and are responsible for the high Km activity, in the
presence of this fraction, of the Enz II F (gf) fraction,
the enzyme II base being completely inactive by itself.
Thus purification of the Enz I Mtl (gf) fraction with
calcium phosphate gel causes a loss of Enz II F (gf)
activity in the absence, but not in the presence of,
a specifier protein such as the Km factor or that
irreversibly bound in the Enz II Mtl (gf) fraction.

A critical factor for the last two models is that
the enzyme II base be constitutive. In the present
experiments this conclusion is suggested by the observa-

tion that in crude extracts the high Km activity, which

218
is constitutive, appears to be replaced to a variable
extent by the lowKm activity in extracts of fructose-
grown cells. It is also suggested by the apparent
conversion of the high Km activity of the Enz II F
(gf) fraction to a LOW'Km system by the Km factor
(Figure 3.17) and by the lack of additivity of the
FlP forming activity of crude extracts. The Km factor
may cause only a partial shift in the Km of the Enz II
F (gf) fraction along with a partial additive effect.
Which type of effect occurs would depend on those
elements which determine the irreversibility of the
formation of the enzyme II complex. The term "Km fac-
tor" with respect to the common enzyme II base model
is probably a misnomer since the system actually involves
a group of specific activators, there being no true modi~
fication of Km values. Recently. R.1N;Walter of this
laboratory has obtained more conclusive evidence (unpub~
lished) for the constitutivity of the enzyme II base.
Addition of preparations containing Km factor prepared
by ultracentrifugation of crude extracts of fructose-
grown cells, to particulate fractions (the enzyme II
fraction) of cells grown on a wide variety of substrates,
produced lowKm systems. However particulate fractions
of mannitol-and sorbitol~grown cells contained high Km
systems which were not modified by the Km factor prepara-

tions. Preliminary evidence suggests that conversion of

219

of the constitutive high Km system to the low'Km system
also occurs in these experiments although this is not a
necessary condition for establishing constitutivity of

the enzyme II base.

At present all of the evidence suggesting a distinc-
tion between the constitutive and the common enzyme II
base models is indirect. The isolation of a second
specifier protein capable of interacting with the Enz
II F (gf) fraction is needed in particular. One
possibility as noted previously is the isolation of a
"high Km factor" from the Enz I Mtl (gf) fraction and
determination of its true specificity by examination of
inhibition patterns. This might lead to identification
of a sugar which would induce a high amount of a revers-
ibly bound factor. Such a procedure, involving the use
of the cumbersome fructose assay with purified, unstable
fractions, might be avoided by a direct attempt to
dissociate more easily assayed enzymes II with treatments
producing solubilization or partial denaturation. Since
PEP systems are involved in transport, that there is at
least some portion of enzyme II which is common for all
PEP systems seems almost certain, since at worst the
membrane itself can be considered a component of the
enzyme II system. From the work of Kaback on the PEP
system in vesicles (100% Simoni gt §l_on enzyme II

sclubilization (98) and other investigations of particulate

220

transport enzymes (8h) it appears that enzyme II fractions
are in fact membrane-associated particles of substantial
size, containing lipid components and perhaps occurring
as vesicles under appropriate conditions. As a rule, in
bacteria genes for inducible enzymes with the same func-
tion occur only once in the genome with few known excep-
tions. Thus it is unlikely that the genes for the poly-
peptides making up the bulk of the structure of inducible
enzymes 11 and‘which are probably responsible for the
common properties of enzymes 11 (binding in the membrane
in a particular orientation suitable for effecting trans-
port, and provision of a site for interaction with HPr)

occur on each substrate-controlled operon.

3.3.3. OTHER MULTI-COMPONENT ENZYME II SYSTEMS. Simulta-
neously with this investigation Simoni, Smith and Roseman
(98) found that it was possible to dissociate two of

the enzyme 11 units of Staphlococcus aureus into sub-

mtl and IIImtl (from the enzyme II

 

units designated 11
for mannitol) and IIgal and IIIgal (from the enzyme II

for galactose). The four components cross react to only

mtl plus 111mt1

Imtl

a slight extent, that is for instance, 11
is active'with mannitol and not galactose, but I
plus 111981 is inactive'with mannitol and galactose.

Thus a specifier protein system, at least one having a
common component for these two enzymes 11 does not appear

to be responsible for these subunits. For the enzyme 11931

221

system a high Km activity was not found in the absence of
111981. The enzyme II for galactose in S, aureus may
also be active with lactose or it may share a common
component with the enzyme II for lactose since mutants
which are probably lacking enzyme 11 activity for lactose
also lack growth on galactose (96).

More recently Kundig and Roseman (101) have found
that a constitutive enzyme II system in E, 3211, which
is reactive with glucose, fructose and mannose, can be
dissociated by solubilizing treatments into (1) three
components which determine the specificity of the three
enzyme II systems, (ii) another protein component which
is required for all three enzyme 11 systems, and (iii)
a lipid component also required for all three systems.
The common protein component undergoes aggregation in
the absence of detergent which is typical of purified
membrane subunits. The specific components appear to
be soluble in the absence of detergent. If the molecular
'weights of the specificity determining components of
this system are found not to be extremely high, this
group of enzymes 11 will have all the properties expected
of a specifier protein system except that in this case

the components are apparently constitutive.

3.3.u. SUPPLEMENTARY DISCUSSIONI—— RELATIONSHIP OF THE
FRUCTOSE ENZYME II SYSTEM TO TRANSPORT. Consideration

of the possible mechanisms for the interaction of the

222

Knlfactor and similar proteins in the enzyme 11 system

is dependent on the relationship between PEP systems

and transport. Since determination of this is presently
incomplete and since there is a proclivity in the field
for rather brief preliminary communications, it is appro-
priate to review the major trends.

The first transport theory to be extensively
developed was the permease theory of Kepes (102)
according to which a permease enzyme,specific for the
substrate,catalyzes the attachment of a substrate
molecule to a carrier protein. The carrier protein
then undergoes a configuration change of some sort
resulting in active transport or facilitated diffusion
depending on the presence or absence of coupling to an
energy source. The experimental evidence for this
theory involved primarily transport systems which are
not at present believed to operate via PEP systems. A
number of binding proteins have been isolated from some
of these non-PEP transport systems (an). All of these
proteins are able to bind substrate specifically in
31353, apparently without the mediation of a permease
molecule. Thus a second carrier mediated transport
mechanism is now possible in which binding to the carrier,
and thus the specificity of thetransport system, is
mediated by the carrier protein itself. In the initial

proposal that PEP systems are essentially a transport

 

223

mechanism Kundig gt El (77) proposed that enzymes 11 were
the carrier component of the system and that it might be
possible to isolate binding proteins from PEP transport
systems analogous to those of nonphosphorylating trans-
port systems. The in vitro manifestation of such binding
proteins would be expected to be that of an activator,
or in the present case a Km factor of some sort, for
enzyme II. Thus it is possible that the specifier
protein proposed here may be a binding protein for fruc~
tose which may or may not bind when dissociated from the
enzyme II complex. If this is the case then the specifier
protein would be implicated as the carrier molecule of
the enzyme II system in analogy with binding proteins
for nonphosphorylating tranSport systems.

It has also been proposed by Tanaka and Lin (78,
103) that the PEP system is not involved in the actual
process of active transport but only in the retention
of substrates. According to this mechanism the sub-
strate is transported across the membrane by facilitated
or nonfacilitated diffusion by a transport system which
may or may not involve enzyme 11. The PEP system, by
phosphorylating the substrate and thus making it non-
transportable, traps it inside the cell so that accumula-
tion or retention of sugar phosphate occurs.

Recently Kaback (100) tested the two theories out-

lined above. It was found that PEP systems can transport

22h

sugars in vitro by causing the accumulation of intramem-‘
branal sugar phosphates in vesicles which form sponta-
neously in purified membrane fractions. It was found
that the membrane-bound PEP system in these preparations
did not phosphorylate sugar which was present inside the
membranes, thus indicating that the PEP system must be
directly involved in the transport process and that it
cannot act simply as a retention mechanism as suggested
by Tanaka and Lin. However the data also indicate that
the_in vitro PEP transport system exhibits none of the

 

characteristics of a carrier mediated mechanism. In
both the absence and presense of PEP, neither facilitated
diffusion of the sugar or sugar phosphate, nor exchange
of extramembranal and intramembranal sugar or sugar
phosphate, was observed. Lack of catalysis of facili-
tated diffusion by enzyme 11 is also suggested by some
properties of mutants lacking enzyme I or HPr. Enzyme

I and HPr mutants of A. aerogenes, strain 1033, do not

 

grow well on glucose at concentrations which are
saturating for the constitutive glucokinase in strain
PRL-R3 (87). Simoni and his collaborators (10h, 105) have
suggested on the basis of sugar uptake data that an
enzyme I mutant of Salmonella typhimurium shows facili-
tated diffusion for<x-methyl glucoside, a substrate for
the glucose transport system. However these mutants do

not grow even at high levels of glucose (10h, 105) and

22S

Kaback (100) found that vesicle preparations from these
mutants did not take up or phosphorylate substrate and
that those from the wild type gave only a low level of
substrate uptake in the absence of PEP, suggesting that
facilitated diffusion, if it occurs, is of a limited
nature perhaps not involving the PEP system. Finally,
the car' (carrier-negative) mutants of Egan and Morse
(88-90) were originally thought to lack a carrier
component because they did not exhibit facilitated
diffusion of galactosides. Subsequently these mutants
have been found to be PEP system mutants lacking enzyme
I (106). This finding invalidated the assay procedure
for facilitated diffusion of galactosides (107-109) and
it has subsequently been suggested that the car" mutant
does show facilitated diffusion (96). However this
conclusion is preliminary and is based on the equilibra-
tion of sugar between extracellular and intracellular
volumes over a period of 15 or more minutes which does
not appear to allow a good discrimination between
facilitated diffusion and the nonfacilitated diffusion
exhibited by this organism for galactosides (88). Further~
more,as previously pointed out by the authors these
mutants do not grow on maltose or sucrose and yet they
have substantial maltase and sucrase activities 12.!1££2
which should allow growth on these substrates if facili-

tated diffusion could be catalyzed by enzyme 11 in the

226
absence of phospho-HPr. In summary, there are increasing

indications that transport via PEP systems may not involve
a carrier component. Facilitated diffusion does occur in
several cases for substrates for which there are PEP
systems, such as glucose in g, 2213, However in this
case Kaback (100) found that the in gitrg PEP transport
system does not show facilitated diffusion and suggested
that other components such as the sugar phosphate phos-
phohydrolases might be involved in the process. As
exemplified by glucose transport in E, 2313 (111), trans-
port kinetics are often very complex, suggesting in most
cases that multiple systems or complex control mechanisms
are involved in the transport of a particular substrate
and militating against the prospect of explaining all
bacterial transport phenomena with a few model systems.
The results of Kaback (101) suggest a transport
theory of P. Mitchel (112) which appears to have been al-
most lost during the development of the carrier theories
for transport via nonphosphorylating mechanisms. For an
active transport system in which there is no alteration
of the chemical structure of the substrates,it is
necessary that there be a carrier mechanism in order to
provide a way for the transport system to interact with-
out causing a permanent chemical alteration in the sub-
strate. However a carrier mechanism is not necessary if
energy can be transduced via a chemical reaction with

the substrate, since in this case there is a configuration

227

change in the substrate itself, so that potentially it
can act as its own "carrier". As conceived by Mitchel
any enzyme has the potential for transporting its sub-
strates via directed diffusion. The substrate in order
to bind must approach the active site from a particular
direction. Once the substrate is converted to product
it would be expected that the product having a somewhat
different binding interaction with the active site
would, except in the case of an unusual active site,
leave the active site along an axis not identical to
that followed by the substrate. If the rotational
freedom of the enzyme is restricted,it is in Mitchells
terms anisotropically oriented with respect to its
environment,and transport relative to the environment
occurs. For enzyme II, a fairly wide angle between the
substrate and product axis, so as to place them on
opposite sides of the membrane, is all that is necessary
for an anisotropic transport system. Such a system
would be unable to catalyze facilitated diffusion or
exchange reactions and in particular, as observed by
Kaback ( HXD), it would not be able to phosphorylate
sugar supplied to the internal side of the membrane
since only the product axis is available from this direc-
tion. Other models can be adapted to the in_ygtrg.trans-
port data; however the correct predictions are unavoid-

able for the anisotrOpic model. Some evidence, possibly

228
indicating that in vivo there is a lack of normal activity

of enzyme II for fructose toward intracellular substrate,

is considered in Section h.2.h.

229

3.h. METHODS

3.h.1. PREPARATION OF EXTRACTS. The methOd‘was different
from that used in Sections 1 and 2 and the same as that
used in Section A. A, aerogenes PRL-R3 was grown in 2,800
mi Fernbach flasks at 32°C with agitation (about 150 cycles/
minute) on the medium described in Section l.h.l. A“500
ml aliquot of fresh mineral medium containing 0.5% of a
given substrate was added to a 500 ml overnight culture
grown on the same substrate, and the culture was incubated
with agitation for about u hours. Cells were harvested by
centrifugation at hPC, washed in cold 0.85% NaCl (approxi-
mately 250 ml/2 g wet wt) and stored frozen for a few days
or used immediately. Unless stated otherwise, further
operations were carried out at O-SOC. Cells were sus-
pended (2.0 ml/l.0 g wet wt) in a 0.1 M Tris-HC1, pH 7. 6,
containing 0.2% mercaptoethanol (unless otherwise indi-
cated), broken by sonication (Section l.h.l) and centri-
fuged at u0,000 x g for 20 minutes to yield a Supernatant
which contained 30-h5 mg/ml protein. Cells‘were grown by
the above procedure even when only enough protein for one

assay was needed. Protein was determined'with the Lowry

assay (1h).

3.11.2. V PURIFICATION OF PEP SYSTEM COMPONENTS.
A. GEL FILTRATION - Sixty to 75 ml of freshly prepared
crude extract were chromatographed on a column (h.80 cm

x about h0.0 cm) containing 650-750 ml of Sephadex G200

 

 

 

230

equilibrated with freshly prepared 0.0h.M Tris-HCl, pH 7.6,
containing 0.2% mercaptoethanol ("Tris buffer"). Tubes
‘were stoppered soon after collection and fractions were
stored in ice water. Where indicated the enzyme I frac-
tions from Sephadex chromatography were pooled and concen-
trated 10 to 20 fold by ultrafiltration through an Amicon
ultrafilter (type UMAl, 10,000 molecular'weight cutoff)
and the same was done for the HPr fractions. In the pro-
cess of concentrating the fractions they‘were diluted with
Tris buffer and reconcentrated so as to obtain at least a
loo-fold reduction of possible low’molecular weight com-
ponents and the old buffer. The pooled fractions used

for the fructose assay included almost the entire range

of the enzyme I and HPr peaks so that when concentrated
enzyme I and HPr fractions were used in the analysis of
enzyme I and HPr elution profiles with the mannitol assay
(as for Figures 3.13.A and B) the pools were subdivided
into front and back portions, so as to avoid cross-conta—
mination of enzyme I and HPr, and concentrated separately.
A small aliquot was taken for the mannitol assays and the
pools‘were recombined for the fructose assays. Enzyme II
pools included a narrow range of fractions from the center
of the enzyme II region and were not concentrated. The
terms Enz I F (gf 10X), Enz II Mtl (gf 1X) etc. refer to
fractions obtained by gel filtration and the degree to

which they*were concentrated. Chromatography of extracts

231

of mannitol-grown cells was repeated 10 times, and of
extracts of fructose-grown cells, h times,with consistent
results for the location of the various activity peaks.
Enzyme components, including crude extracts, were stored
at 0°C on ice. Gel-filtered components were used in
assays as soon as possible, generally within two weeks,
and old enzyme II fractions were not used in the fructose
assay even though no changes were observed with aging.

B. HEAT TREATMENT OF HPr FRACTIONS - Concentrated HPr
fractions from gel filtration were heated for 10 minutes
in a boiling water bath and centrifuged at h0,000xg for

20 minutes to remove precipitated protein.

C. CALCIUM PHOSPHATE GEL PURIFICATION OF ENZ I Mtl (gf) -
For the experiment given in Table 3.9, 2.5 ml of a 20-fold
concentrated Enz I NR1 (gf) fraction was diluted to 32.5
ml'with Tris buffer, 3.2 ml Of a suspension of CaPOh gel
(62 mg/ml of suspension) wms added, the mixture was stirred
for several minutes and the gel was removed by centrifuga-
tion for 5 minutes at 20,000xg. The gel'was eluted‘with
32.5 ml of 0.05 M and 0.10 M sodium phosphate (pH 7.5)
containing 0.2% mercaptoethanol. The fractions were
assayed with the mannitol assay for enzyme I and the 0.05
M fraction, containing 83 percent of the original activity,
was repeatedly ultrafiltered and diluted with Tris buffer
so as to give a 99.9% replacement of phOSphate buffer

with Tris buffer and to produce a final fraction containing

232

the same amount of enzyme I activity per ml as the original
Enz I Mtl (gf 20X) fraction. About 5-fold purification in

the calcium phosphate gel step was obtained.

3.b.3. ASSAY FOR PEP-DEPENDENT CONVERSION OF FRUCTOSE TO
FRUCTOSE-l-PHOSPHATE (FRUCTOSE ASSAY). The incubation
mixture contained (in the order of addition): no mM Tris-
HCl, pH 7.6, (no mercaptoethanol), 0.05 mM MgC12, the PEP
system components, 1.0 to 300 mM fructose and 6.0 mM PEP
(tricyclohexyl-ammonium salt). For assays of crude
extracts 0.2 m1 of an extract containing 30 to A5 mg
protein/ml was used per ml of incubation mixture. Higher
amounts were used in some cases (see Table 3.u) and seemed
to give higher Km factor index values. The recommended
amount would be about 10 mg protein of crude extract per
ml of incubation mixture. For typical amounts of gel-
filtered PEP system components see the legends to the
figures and tables. The amount of enzyme I and HPr used
may be considerably more than is necessary. The level of
enzyme components in the assay was adjusted so as to ob-
tain approximately a final concentration of FlP in the
incubation mixture of 0.0001 to 0.0005 M although in a

few cases using crude extracts the final concentration

'was as high as 0.003 M. The assay was made at a variety
of total volumes of from 1.0 ml to 0.5 ml in small plastic

centrifuge tubes and at 0.2 ml in 6 x 50 cm glass tubes.

233

The mixture was incubated for 10 minutes at 25°C after which
the reaction was stopped by heating at 100°C for 6 minutes
and protein was removed immediately by centrifugation.
Glass tubes were centrifuged for 20 to 30 minutes at 16,000
xg and the supernatant was decanted by holding the tube up-
side down at a LLSDangle, injecting air bubbles with a
syringe_and catching the drop in a small test tube. The
supernatants were stored at -2OOC. The end-point assay
used for FlP determination contained in a final volume of
0.15 ml: glycyiglycine, pH 7.5, 20 umoles,DPNH 0.007
pmole, MgC12 1.0 pmole, ATP 0.5 pmole, sample 0.05 ml
(usually),excess asglycerophosphate dehydrogenase, and
excess rabbit muscle aldolase. The reaction was initiated
‘with the addition of 0.02 ml of fructose-l-phosphate kinase
purified up through the calcium phosphate gel step and con-
taining 0.1 M sodium phosphate, pH 7.5, (about 70-fold
purified, see Section 1.2.3). FDP was assayed by
initiating the reaction with aldolase rather than FlPK.

The O'D'BhO was monitored continuously. The assay passed
through a moderately sharp inflection point when the F1?
was completely utilized and then continued at a much reduced
linear rate. The shape of the inflection point due to
exhaustion of FlP is distinctive. .Any deviation of the
shape from that obtained using a known sample of FlP was
cause for concern, generally indicating that DPNH had
become limiting. In order to obtain the initial and final

2311

points in the utilization of FlP the initial and final por-
tion of the 0.D. traces were extrapolated back to zero time.
The assay produced one mole of DPN per mole of FlP or FDP

and‘was linearly proportional to FlP concentration.

3.u.u. ASSAY FOR PEP-DEPENDENT CONVERSION'OF MANNITOL TO
MANNITOL-l-PHOSPHATE (MANNITOL.ASSAY) - The assay cuvette
contained in a volume of 0.15 ml (in the order of addition):
133 mM glycylglycine (pH 7.5), 0.667 mM TPN, 0.33 mM DPN,
excess glucose-6-phosphate dehydrogenase, excess mannitol-

l-phosphate dehydrogenase, 6.67 mM MgC12, enzyme II for

mannitol and other PEP system components, 3.33 mM mannitol
(unless noted otherwise), 3.33 mM PEP and 3.33 mM ATP. The

O.D. 'was monitored continuously. Typical amounts of con-

0
centigted PEP system components are given in the legend of
Figure 3.13. Five-fold less enzyme I and HPr from unconcen—
trated fractions could be used with equivalent results. The
amount of the limiting component was kept as IOW’aS possible
[0.001 ml of Enz II Mtl (gf 1X) for instance] and initial 1

rates were taken. Blank values for cross contamination of

fractions were subtracted'when necessary.

3.h.5. PURIFICATION OF MANNITOL-l-PHOSPHATE DEHYDROGENASE.
The procedure is a modification of that of Liss et 31 (7).
For a typical purification,l6 ml of a crude extract of
mannitol-grown cells was prepared in 0.1 M Tris-HCl, pH

7.6, containing 0.2% mercaptoethanol as described in Section

235

3.h.l. 1.6 ml of 10% protamine sulfate (pH 7.0) in water
was added slowly with stirring and the mixture was stirred
for 15 minutes and centrifuged at h0,000xg for 10 minutes.
15 m1 of the supernatant was adjusted to pH 5.0 with dilute
acetic acid, 60 m1 of CaPOu gel (21 mg/ml of suspension)

was added, the pH was readjusted to pH 5.0, (the pH meter
was calibrated at 25°C, the pH measurement was made at 0°C),
the mixture was stirred for 10 minutes and centrifuged for
5 minutes at 20,000xg. The gel was suspended in 0.0h M
Tris-HCl, pH 7.6, adjusted to pH 8.0 with 1 N NaOH and cen-
trifuged at 20,000xg for 10 minutes. The procedure gave
about uo-fold purification with about 50% recovery. The
preparations contained no DPNH oxidase activity but had a
small amount of HPr activity. The enzyme retained useful
activity for about three‘weeks stored at 0°C. The assay
for mannitol-l-phosphate dehydrogenase contained in a volume
of 0.15 ml: 133 mM glycylglycine (pH 7.5), 0.h7 mM DPNH,
6.67 mM'F6P and mannitol-l-phosphate dehydrogenase prepara-
tion. The O-D-Buo‘was monitored continuously. Mannitol-1-
phosphate dehydrogenase is separated from the Enz I Mtl (gf)
fraction during DEAE cellulose purification and from HPr Mtl

(gf) fractions during heat treatment.

3.h.6. ASSAY'FOR ENZYME II FOR MANNOSE. The assay cuvette
contained in a volume of 0.15 ml: 133 mM glycylglycine,
0.667 mM'TPN, excess glucose-6-phosphate dehydrogenase,

 

236

excess phosphomannose isomerase, 6.66 mM'MgClg, 0.01 ml
Enz I F (gf 20X), 0.01 ml HPr F (gf 20X heat treated)
limiting the Enz II mannose (gf 1X) and 3.33 mM PEP.
The Enz II mannose (gf 1X) was obtained from the enzyme
II region of the elution profile of a crude extract of
mannose-grown cells chromatographed on Sephadex 6200.
The phosphomannose isomerase was prepared by D. Allison

according to the method of M. Y. Kamel (112).

 

 

 

SECTION II. MUTATIONAL ANALYSIS OF FRUCTOSE METABOLISM

SUMMARY: The isolation of mutants of A, aerogenes exhibiting
impaired metabolism of fructose specifically.resu1ted in

two types of mutants. One type lacked the low Km phospho-
enolpyruvate-dependent phosphotransferase system and the

other lacked fructose-l-phosphate kinase activity. Charac-

. _ .__.q

terization of the two types of mutants indicated that the
Km factor and fructose-l-phosphate kinase are involved in 1
the in 3133 metabolism of exogenously supplied fructose.

Participation of the hexose phosphateihexose (H20) phospho-

transferase in fructose metabolism could not be substan-

titated. Also described are the results of collaborative

investigations of mutants lacking 6-fructokinase, fructose

diphosphate phosphatase, and fructose-6-phosphate kinase.

These investigations establish the role of 6-fructokinase

in sucrose metabolism and the £2.!£!2 relationship be-

tween the fructose-l-phosphate and fructose-6-phosphate

pathways. They have also indicated the relationship of

the fructose-l-phosphate pathway to the gluconeogenic

and glycolytic pathways. The preliminary analysis of a

mutant lacking aldolase activity is also described.

h.l. INTRODUCTION
The isolation of mutants was initiated in order to
evaluate the in vivo significance of the hexose phosphate:

hexose (H20)phosphotransferase (Section 2) to fructose

237

 

lilulaumhuflnllai it .n JV

 

238

metabolism, to eValuate the in viva significance of fruc-
tose-l—phosphate kinase and to clarify the relationship
of the FlP pathway to the F6P pathway for fructose metab~
olism. Since no method was available at the time for the
isolation of carbohydrate-negative mutants of A. aerogenes,
a procedure was developed. The procedure is less convenient
technically than other methods now known. However it was
sensitive enough to detect mutants whiCh, though exhibiting
rather minor impairments of growth on fructose, were quite
important to an analysis of fructose metabolism. Subse—
quent to the preliminary analysis of the mutants obtained,
more detailed and expanded analyses were carried out in
some cases by other personnel in this laboratory. In
such cases the results are summarized, additional data
if any are presented and the problems are discussed at
their present stage of development.

Portions of the material presented in this sectibn

are covered in references 13, 92, 9h and 95.

 

239

h.2. RESULTS AND DISCUSSION

h.2.l. TECHNIQUES FOR MUTANT ISOLATION. The basic proce-
dure for the isolation of mutants included: (i) ethyl
methane sulfonate (EMS) treatment, (ii) expression of
mutants and dilution of undesirable mutants, (iii) peni-
cillin selection, (iv) selection on indicator agar, (v)
growth testing on agar containing various sugars, and (vi)
growth tests in liquid media. The specific isolation pro~
cedure for each mutant is given in Methods. Some observa-
tions on these procedures follow.

About one—third or so of EMS-treated cells, when grown
on any substrate for three generations and plated on glucose-
mineral agar, produced colonies smaller than that of the wild
type. It was observed (phase contrast microscopy) that after
treatment with EMS and a few generations of growth, many of
the cells were filamentous. The proportion of filamentous
forms decreased with the number of generations of growth in
glucose-mineral media and at 5 generations only a few
were present. However these few persisted for up to 20
generations. Two other forms observed in low numbers
after EMS treatment were branched forms (usually Y-shaped)
and bloated forms (usually club-shaped). Both of these- ‘
types also persisted for many generations. Of the mutants
finally isolated, none were filamentous: however two of them
produced branched and bloated forms (mutants 010 and O8).

Penicillin selection was usually made in the presence

 

 

 

2110

of fructose with cell concentrations of about 2 x 108 to

l x 109 cells per m1. For expressed, EMS-treated cells,
at 200 units of penicillin per ml, survival was about 1

X 10-2 and at 2,000 or h,000 units per m1 it was 1 x 10-3

to 1 x lO-h. For penicillin selection the cultures were
incubated until the cells were actively growing at which
point penicillin was added. In several experiments in
Which the preincubation was not used only a few highly
pleiotrOpic mutants were isolated. For every experiment
the progress of the penicillin treatment was followed by
phase contrast microscopy. Samples were taken for mutant
isolation from the point where survival appeared to about
10% and thereafter for about 3 hours. It is believed that
by doing this loss of mutants due to cross-feeding, which
wms probably severe at longer treatment times, was avoided.
It was observed that for samples taken in the time period
used, the range of colony size on any type of agar was
quite‘wide, indicating survival of a variety of different
types of mutants. However for samples taken 2 to 5 hours
beyond this period the range in colony size became
increasingly narrow, indicating a loss of mutants and,
most likely, the eventual predominance of mutants resistant
to penicillin.

The indicator agar used in most experiments contained
0.5% fructose and 0.005% of another carbohydrate (usually

glucose). This agar was intended to give small colonies

2A1

for fructose-negative mutants. The colony diameter picked
for further testing ranged from very small to about one-half
that of the wild type. It was observed that the very
smallest colonies picked were generally unable to grow on
nutrient agar. In the last series of experiments fructose-
EMB agar was used. The variation in colony types of
penicillin-treated cells on EMB agar was extremely wide. Gen-
erally the lighhnhcoiored colonies were picked. However in
some experiments any colony which varied in the slightest 1
way from the wild type was selected and from the results
of these experiments given below (Section h.2.6) it appears
that use of EMB agar in this way provides primarily an
exclusion of the wild type rather than a selection for
lack of growth on fructose. It may be that slight differences
in metabolic control patterns cause variations in fermenta-
tion product composition which give detectable changes on
EMB agar.

The carbohydrates used for preliminary growth testing
on agar (and also for expression) were fructose, glycerol,
glucose, mannose and mannitol, an unfortunate combination
since the metabolism of all of these carbohydrates was
subsequently found by Tanaka and Lin (87) to be affected
by the loss of enzyme 1 or HPr, thus precluding the isola-
tion of such mutants in these experiments. Following this
preliminary growth testing, mutants were tested in liquid
media. Considering the apparent technical simplicity of

2&2

growth testing in liquid media the results were surprisingly
variable, even occasionally for the wild type. The condition
of the inoculum,in terms of how long it had been stored and
at what temperature,appeared to be especially important in
determining the lag period before initiation of growth on
those substrates which are metabolized at substantially less
than the maximum rate by the wild type. For the mutants,
growth on such substrates tended to be the most severely
affected whenever there were indications of pleiotropic
effects. Another source of variation in the growth of
mutants may be due to the fact that none of the sugars are
absolutely free of other carbohydrates, particularly after
sterilization. The presence of these impurities may cause
lags in the growth of mutants for which the impurities are
toxic. A possible example of this type of effect has been
observed in an E, 3211 mutant for which glucose is toxic
(22. 113).

About 2,000 colonies suspected of being fructose-nega-
tive‘were isolated from indicator agar and tested in
patches on agar. About ho mutants negative only on fructose
‘were found but about half of these were lost, presumably
due to rapid reversion back to the wild type or secondary
revertant types. The growth curves and other information
on the mutants to be presented are given in Figure h.l
and the results of the enzymatic analysis of these mutants

are given in Table u.1

2143

In interpreting enzyme levels in mutants a number of
factors must be considered, some of‘which may be obvious.
The activity found in crude extracts of wild type cells
may include minor rates catalyzed by enzymes other than
that for which the assay is intended so that it is difficult
to Judge the significance of very low activities found in
mutants. Besides multiple enzymes with similar reactivities,
residual activities could be due to the slow catalysis of
completely different reaction sequences than that for which
the assay is intended and to activities dependent on conta-
minants in the reagents. For many types of loss of enzyma-
tic activity it is very difficult to separate cause from
effect. Considering the semianaerobic conditions used for
growth, for cells grown on substrates for which they exhibit
impaired metabolism the slower growth may result in greater
aeration, causing a general depression of activities com-
pared with the wild type (See Section 1.2.3). General
perturbations of enzyme levels can also be expected from
loss of a constitutive enzyme in a pathway for carbohydrate
degradation since these pathways are probably involved in
complex and far-reaching control systems. In fact, as is
suspected for FlPK, loss of an apparently inducible enzyme
may cause changes in constitutive metabolism. For consti-
tutive membrane-bound enzymes, the possibility that a loss
of activity is due to an indirect effect of a lesion in

membrane structure outweighs any other consideration,

 

21:11.

unless another rationale is strongly supported by other
evidence. For inducible enzymes there is no simple way
to differentiate absolutely between a lack of induction
and a lesion in enzyme structure. Some of the assays

in Table h.l were repeated a number of times with slight
variations in growth conditions in hopes of making in-
direct effects apparent. These manipulations did result
in variations in some cases although not necessarily

due to the intended variable.

2115

Figure h.l. GROWTH CURVES OF MUTANTS. Cells were grown
in 7 ml test tube cultures at 32°C with shaking and 0.D.
was measured at 600 nm with a Coleman Jr. colorimeter.
Media used included: (1) mineral medium plus 0.5% of
various substrates as indicated, (2) to detect fruc-
tose toxicity or other effects - mineral medium plus
0.5% of one substrate and 0.05% of another substrate

as indicated and (3) to test for possible permeation
effects - mineral medium plus 1.0% fructose. Cells for
inoculation were grown overnight in the indicated media
and were not washed before inoculation. A 0.1 ml inocu-
lum was used.. Blanks for the wild type containing no
substrate gave no detectable growth. The dotted lines
in some of the figures give the 0.0. plot for the growth
of the wild type on glucose in the same batch, using an
inoculum grown on the same substrate. See Methods for
the isolation and properties of the bacteriophage

referred to in several figures.

Figure h.l.A.1. WILD TYPE, PRL-R3. Inoculum grown

on nutrient broth. These curves are the controls for

those in Figure h.2.B.

 

2116

Figure Ll’olvolo

 

F l I I

I
at A. WILD TYPE glucose plus
0.0 % ructose ,

o7 '- f
glucose '
:2. .6 - glycerol K/Oé
:fffggg
:3 .5 __ fructose ;;;;;;;. ’,,,’/’:::;77‘
,. sucrose ///////f:::;; ///////

:jjjj;2__zxylose
L-

arabinose

. 0......

O-D-6OO

  

galactose

.1 —

 

I l I l l

 

 

O 100 200 300 400 500
TIME (min)

 

2117

 

Figure h.1.A.2. WILD TYPE, PRL-R3 . Inoculum grown
on glucose. The rate on glycerol is lower, and the

lag with ribose and xylose shorter, than with an

 

inoculum of nutrient broth-grown cells (Figure h.l.A.lL

 

 

   

 

Figure h.1.A.2

0.130600

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I ___./:
g/._.:/ /;/. ./ . L-Tylose
‘ n “‘r‘>I./.I——I.7rt—I——I===I=.I=.___
0 100 200 300 400 500 700 900 1100 1300

TIME (min)

 

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Figure b.l.1. MUTANT CC7. Inoculum grown on nutrient
broth. Growth on agar: a lag period was observed on
all substrates except nutrient agar, grows on sucrose,
very negative on fructose. The lag period varied some-
what in different experiments. This mutant is similar
to a number of others isolated although it is distin—
guished by the degree to which growth on fructose is

impaired relative to that on other carbohydrates.

 

 

 

Figure ”'LI'

258

 

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Figure h.l.K. MUTANT 012. (Not included in Table h.l)
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grown on glucose. Cells gave typical plaques when

tested with phage.

262

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270

4.2-2. MUTANTS LACKING THE LOW Km PEP SYSTEM FOR FRUCTOSE.
The two mutants in Table h.1 which are designated as

‘ lacking the Km factor, 0017 and RR15 (also BBBB6Gy)

showed a relatively small decrease in the growth rate on
fructose and a lack of toxicity of fructose for growth

on glucose. All were able to induce FlPK to a reasonable
level, indicating that the induction mechanism for this
enzyme and presumably the Km factor is functional. All
three mutants were able to produce the elevated high Km
activity for fructose found in mannitol-induced cells
indicating that other components of the PEP system are
functional. When induced on fructose the mutants exhibited
the less active high Km PEP system that is observed using
purified components from fructose-grown cells in the absence
of the Km factor, and which is probably identical to the
constitutive PEP system for fructose. 0017 is the simplest
of these mutants, having the highest level of FlPK, complete
absence of the lowKm PEP system, a normal level of 6-FK
and a growth pattern without a lag period and without
pleiotropic effects. RR15 showed an occasional decrease in
the level of FlPK and an occasional increase in the level
of 6-FK. The latter effect is typical of mutants lacking
FlPK.(see below). BBB36Gy gave a definite lag period in
guwnvth, exhibited a certain degree of pleiotropic depression

of growth rates and in one case had some lowKm PEP activity.

 

271

R. W. Walter of this laboratory has found (unpublished) that
the growth rate of 0017 on fructose is concentration depen-
dent, having a "Km" of about 0.01 M. This may explain the
fact that in testing the growth of these mutants on fructose-
agar the lack of growth was more distinctive than the growth-
curves might suggest, particularly that for RR15.‘ Since
diffusion of substrates in agar (as indicated by diffusion
of dyes in several experiments - also see reference 22) is
rather slow; cells growing on agar are probably exposed to .
a lower concentration of substrate than those in mineral
medium. However fructose-EMB agar and fructose-MacConkey
agar did not distinguish RR15 from the‘wild type.

The effects of various components of the PEP system
added to crude extracts of 0017 and RR15 (Table h.2)
indicated that at least for 0017 the missing component.
is the Km factor and that the other three components are
not involved in the lesion. For 0017, as observed with
gel-filtered components (Section 3.2.8), a two~fold activa-
‘tion as well as an increase in the Km factor index occurred
Iwith addition of the Km factor. The amount of Km factor
added.in this experiment resulted in a final concentration
IMhich was probably 50-100% of that present in the assay
of the‘crude extract from the wild type (as calculated
from the dilution involved in gel filtration, assuming
conuilete recovery of Km factor activity). The increased

index value obtained for 0017 was in the same range as

272

 

 

 

 

 

 

 

 

 

 

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273
that for crude extracts of fructose-grown cells. Note that
the‘wild type also gave a slightly higher ratio in the.
presence of the Km factor, indicating perhaps a lack of
saturation with Km factor due to dilution of the crude
extract in the assay. RR15 gave only a partial increase
in the index value in the presence of the Km factor and
did not show activation at 0.2 M fructose. A possible
explanation is that the response is partially blocked by
the existence of a defective Km factor.

A further test of 0017 is shown in Figure h.2. A
crude extract of fructose-grown wild type cells was chromato-
graphed on Sephadex G200. Aliquots of the fractions were
added to a crude extract of fructose-induced 0017 cells
and the mixture was assayed with the fructose assay at 0.2
M and 0.002 M fructose. From left to right in Figure 4-2.
there is first a peak in activity at 0.2 M fructose in the
enzyme 11 region without a corresponding peak in activity
at 0.002 M fructose. This is due to the addition of the
Enz II F (gf) fraction from the elution profile to the
unimpaired enzyme I-HPr system in the 0017 extract. This
peak is followed by a peak in both the 0.002 M and the 0.2
M activity which is due to the effect of the Km factor
fraction on the Enz II F component in the 0017 extract.
Note that the position of the peak in the Km factor index
values with respect to the enzyme I peak corresponds very

‘well with the peak obtained using the Km factor assay which

 

 

 

27h

Figure h.2. GEL FILTRATION OF A CRUDE EXTRACT OF FRUCTOSE~
GROWN CELLS - 0017 ASSAY. As described for Figure 3.13, a
crude extract of fructose-grown wild type cells was chroma-
tographed on Sephadex G200 and assayed for enzyme I (A)

and HPr (n) using the mannitol assay. The fructose assay
(Section 3.h.3), 0.2 m1 total volume, containing 0.0M m1

of a crude extract of fructose-grown cells of mutant 0017

and 0.1 m1 aliquots of the fractions, was run at 0.2 M

 

fructose (O) and 0.002 M fructose (a).

 

 

LCETTEXET
ibed for figs}
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, containing
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,

).

FRUCTOSE ASSAY (umoles FIP/min/assay)

Figure 4.2.

.1

 

ENZYME I and HPr (umoles TPNH/min/ml)

.5

.1

275

 

 

 

 
  
 
 

 

 

 

VOLUME (ml)

I l l
. .6
Enzyme I
_ .5
.2 M Factor
activity \/ Index .15,
_ -3
_. .2
01:;
..\
\
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_ no.002-14 .1
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I
0 200

Km FACTOR INDEX

276

was dependent on purified components (Figure 3.13).

The behavior of 0017 in particular, substantiates
first, that the fructose PEP system is the in 3113 source
of FlP in fructose metabolism. Second, it indicates that
the Km factor is necessary for the normal functioning of
the PEP system. The apparent inability to isolate mutants
defective only in fructose metabolism and lacking the high
Kulfructose PEP system, even though such mutants might be
easier to detect since they‘would presumably lack all
ability to transport fructose and to produce FlP, is
consistent with either, (i) the enzyme 11 base, as mani-
fested by the high Km constitutive activity, being involved
in the metabolism of more than one carbohydrate, or (ii)
that the constitutive PEP system is not involved in fruc—
tose metabolism in any way. However as presented in Section
3.3.2 there are several lines of evidence supporting the
involvement of a constitutive component in enzyme II activ-
ity. According to the common enzyme II base model (Section
3.3.2) one should be able to isolate pleiotropic mutants
lacking enzyme II activities. The phenotype for such
mutants is impossible to predict but it need not be the
same as that of enzyme I and HPr mutants. The finding of
‘Tanaka and Lin (85) of a mutant lacking growth only on
mannitol and lacking the enzyme II activity for mannitol

does not contradict the involvement of an enzyme II base

277
in this system since the assay used in these experiments
was such that there could not have been a mannitol
specifier protein present in the assay components used,
so that the assay would have been negative either for
loss of a specifier protein or of a conventional enzyme
II. It is necessary in fact to predict, due to the
mannitol inhibition effect (Section 3.2.7),that in parti-
cular the enzyme II for mannitol would be lost as well
as that for fructose due to a lesion in the enzyme 11
base. Another enzyme 11 mutant which has been reported
is the "cnc" mutant of Staphlococcus aureus which
probably lacks an enzyme 11 activity which is necessary
for the metabolism of both lactose and galactose (96).
Since it is the galactose moiety of lactose which is
phosphorylated in the metabolism of lactose in this
organism (107, lOBL this enzyme II may simply have a
dual specificity.

 

278
n.2. 3. HEXOSE PHOSPHATE:l-iBXOSE (H20) Pl-DSPHOTRANSFERASE
LEVELS OF PLEIOTROPIC MUTANTS. The most significant re-
sult concerning the hexose phosphate:hexose (H23) phospho-
transferase (the H-phosphotransferase, Section 2) was that
no mutants defective only in growth on fructose were found
to lack the FlP,G : GP activity catalyzed by this enzyme.
Two mutants, 010 and 08, were found which have low levels
of the enzyme (Table u.l). However they do not support the
proposal made in Section 2.3 that-the H-phosphotransferase
is involved in a biosynthetic pathway requiring glucose.
Although both are glucose positive pleiotropes neither grows
‘weil on sucrose. Furthermore 08 (as seen in Figure h.l.H)
and 010 did not show increased growth on fructose when sup-
plemented with glucose. Both mutants had low levels of most
of the enzymes assayed suggesting that the decreased level
of the H-phosphotransferase was due to indirect effects.
The H-phosphotransferase activity found in Q10 (7%) was not
significant under the particular conditions of the assay;
that in 08 (16%) was significant insofar as the accuracy
of assay‘was concerned. However the assay is of the type
mentioned above (Section h.2.l) that is likely to give low
rates using crude extracts in the complete absence of the
enzyme for which it is intended, due to various reactions
catalyzed by other enzymes. The abnormal morphology of
(310 and 08 (see the legend of Figures h.l.G and H) may be

due to a direct or indirect effect on cell wall and/or

 

 

279

membrane structure. Since the H-phosphotransferase could
be removed by the EDTA-osmotic shock procedure (Section
2.2.h) this suggests that perhaps the enzyme was lost in
the mutants due to lack of the proper binding conditions
in the periplasmic area. Although it is impossible to
come to any conclusion concerning Q10 and 08 it is never-
theless peculiar that such low levels of an enzyme, the
in vitgg properties of which suggest some sort of involve-
ment related to glucose metabolism, should occur in
pleiotropic mutants which grow well on glucose in parti—
cular. The only way to establish that loss of a consti-
tutive membrane-bound enzyme is due to a structural
lesion in the enzyme would be to obtain electrophoretic
or other variants of it from second-site revertants of
the mutant. The lack of correlation between GlP phos-
phophatase activity and FlP,G : GP activity suggests

that the H-phosphotransferase may not contribute as much
to the total phosphatase activity in crude extracts as
its 5:1 phosphatase to hexose phosphotransferase ratio

in purified fractions would suggest (Section 2). There
was also a lack of correlation, particularly for Q10,
between the H-phOSphotransferase activity and the activity

of the constitutive PEP system for fructose.

 

F i_

280
h.2.h. MUTANTS LACKING FIPK. Mutants DD31 and CCC38,
which were isolated independently of each other, gave al-
most identical growth patterns, exhibiting slow growth on
fructose and toxicity of fructose for growth on glucose.
Both mutants lacked FIPK, had a constitutive low Km PEP
system for fructose, and an elevated level of 6-FK. An
extensive analysis of these mutants, in which I participated
only sporadically, resulted from an investigation carried
out by N. E. Kelker, of this laboratory, the initial objec-
tive of which was the evaluation of the role of 6-FK in
metabolism. However the investigation has expanded so as
to involve every aspect of fructose metabolism. The first
phase of this work (13) will be presented briefly.

From DD31 which grows slowly on fructose and thus
produces red colonies on fructose—MacConkey agar, an EMS
induced mutant lb, was isolated which gave pale colonies.
Mutant 1h grew very slowly on fructose and also did not
grow on sucrose. To a somewhat greater extent than in the
case of DD31, fructose was toxic for the growth of in on
glucose. From In a spontaneous revertant, 1h RAF, was
isolated on fructose-NbcConkey agar. 1h RAF grew on both
fructose and sucrose at the wild type rate. The enzymatic
characterization of these mutants is given in Table h.3.
Mutant DD31 lacked FlPK and had a constitutive PEP system.
Mutant 1h is a double mutant lacking 6-FK as well as FlPK

and having a constitutive PEP system. The revertant, 1h RAF,

 

281

still lacked 6rFK but had reverted so as to regain the
activity of FlPK. The low Km PEP system in IA RAF was
induced by fructose or sucrose but not by glucose. An
analysis of the accumulation patterns of various inter-
mediates in these mutants is given in Table u.h. The
lack of variation with respect to time in the FlP and
HJP levels for the wild type and DD31 (see legend of
Table h.h) indicate that the values represent steady
state levels or, possibly in the case of FlP accumula-
tion in DD31, a static level. This is in accordance
with results obtained under similar conditions with E.
3311 (11h) which indicate that steady state levels of
sugar phosphates and other intermediates are established
within about one to two minutes after exposure to sub—
strate. For mutant 1h in the presence of only fructose
the attainment of a steady state or static level of F1?
probably requires more than five minutes (see legend of
Table h.h).

The analysis of this series of mutants is based on
the following hypothesis (which is consistent with the
results described in Section I and 3) for the metabolism
of fructose and sucrose and its control. The actual
inducer of the Km factor and FlPK is FlP, the only
intermediate unique to fructose metaboliSm. The
6-FK and sucrase are located on two different operons
which are induced by the presence of intracellular fruc-

tose. The 6-FK (as well as sucrase) is not involved in

 

 

282

 

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287

the metabolism of exogenously supplied fructose but in the

metabolism of intracellular fructose which is produced in

the degradation of carbohydrates containing fructose. In

Figure h.3 are given the proposed pathways for fructose

metabolism and other pathways in this area of metabolism.

It is assumed for the purpose of this discussion that sucrose

is not transported via a PEP system. This is not neces-

sarily the case. ‘It is quite possible that the glucose
moiety of sucrose is phosphorylated during transport and
the sucrose phosphate is the actual substrate for sucrase

(which has not been characterized). This situation would

not alter the following discussion.1 With this model our

rationale for mutant DD31 and its derivatives in accordance
with the data in Table 4.3 and h.h is as follows.

A. Mutant DD31. The lack of FlPK in DD31 causes FlP
produced by the PEP system to accumulate to a level
which is about 20 times higher than normal (Table h.h),
thus causing the toxicity of fructose during growth
on glucose. There are two mechanisms which might be
responsible for the slow growth of DD31 on fructose.
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to free intracellular fructose by either an unknown
phosphatase, hexose phosphotransferase, or other
reaction. This conversion of FlP to fructose would
not occur at a significant rate in the wild type in

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290
there may be a mechanism, not necessarily operative in the
wild type, by which free fructose is transported across
the membrane. The increased level of intracellular fruc-
tose in DD31 causes 6-FK to be induced to a level which
is higher than that in wild type cells growing on fruc-
tose. (This is more evident in Table u.l.E than in
Table h.3.) The fructose is phosphorylated by the ele-
vated 6—FK producing F6P, the further degradation of
‘which allows DD31 to grow slowly on fructose. The loss
of FlPK causes the PEP system to become constitutive
due to endogenous induction. That is, in the wild type
during growth on glucose or other substrates FlP forms
at a very low rate due to unknown reactions involving
perhaps the nonspecificity of various enzymes. In the
wild type this FlP is removed by the constitutive (or
basal level of FlPK. However in DD31 it accumulates
and causes the low Km PEP system to assume constitu-
tivity.
Mutant 1h. Loss of 6-FK in mutant in causes loss of
the slow growth on fructose as well as loss of growth
on sucrose and other sugars containing fructose. The
increase in fructOse toxicity is a minor effect which
cannot be explained at present. If, as proposed for
DD31, accumulation of FlP results in an irreversible
hydrolysis producing fructose, this might be responsible
for the increased toxicity observed for mutant 1h. The

PEP system and the hydrolytic reaction would constitute an

 

291

apparent PEP phosphatase which would result in a loss of

PEP, an accumulation of intracellular fructose and pos-
sibly an accumulation of inorganic phosphate and pyruvate,
any one of which might cause increased inhibition. The
lack of growth of mutant In on the glucose moiety of
sucrose is apparently due to the toxic effect of the

FlP which accumulates during utilization of sucrose

(Table h.h). This accumulation of FlP is probably due

to the excretion of accumulated fructose which reenters 3

 

the cell as FlP via the constitutive low Km PEP system
present in this mutant.

Mutant 1h RAF. When FlPK is regained in In RAF the
mutant now grows completely normally on fructose even
though it lacks 6-FK, thus indicating that this enzyme
has at most a minor role in the normal metabolism of
extracellular fructose. The reason for this appears
to be that fructose is transported into the cell as
FlP rather than free fructose so that there is not
enough substrate available to saturate 6-FK. The
regain of FIPK in 1h RAF causes the level of F1? in
cells utilizing fructose to drop to the normal concen-
tration and it also stops the induction of the IOW'Km
PEP system during growth on glucose since endogenously
formed FlP is removed by the constitutive level of FlPK.
However during growth on sucrose, 1h RAF presumably

accumulates fructose due to the lack of 6-FK. The

292
fructose is probably excreted and metabolized via the
FlP pathway in the same way that exogenously supplied
fructose is metabolized, thus resulting in the induc-
tion of FlPK and the Km factor and in the accumulation
of FlP during growth on sucrose to a level similar to
that observed during growth on fructose (Table h.h).
The reason that we propose that intracellular fructose
for the 6-FK-dependent growth of mutant DD31 may be derived

from FlP (instead of the more obvious possibility that it

 

comes from the external medium by a mechanism which allows
free fructose to pass through the membrane) is that analysis
of an FDPase-negative mutant indicates that 6-FK is com-
pletely nonfunctional in the metabolism of exogenously
supplied fructose (Section h.2.6). The lack of effect of
the loss of 6-FK in IA RAF on the rate of fructose metabo-
lism is consistent with this hypothesis. However it is not
considered to be supporting evidence since carbohydrate me-
tabolism is not necessarily the rate-limiting step in normal
growth, so that loss of a minor component of the capacity for
fructose metabolism might not cause a change in the growth
rate. Since extrapolation of results derived from the
analysis of the FDPase mutant to the present situation is

a rather indirect procedure, we cannot eliminate the pos-
sibility that free fructose is transported across the mem-
brane in the case of mutant DD31. It could be proposed that
this permeation is an abnormal property which is not opera—

tive in the wild type. Since it appears that in the case of

293
at least 1h RAF, fructose can be excreted during the metab-
olism of sucrose, the same permeation mechanism might be in-
volved in the growth of DD31.

The endogenous induction hypothesis has been applied to
galactose-negative mutants of E. soil which lack galacto-
kinase and therefore accumulate galactose so that the epi-
merase and transferase become constitutive (117, 118). In
this case the source of endogenous galactose, as determined —

by mutational analysis, is known, although endogenous galac-

tose has not been detected by direct assays (117, 118). One

 

 

 

possible source of endogenous FlP is a slow hydrolysis of
FDP at the 6 position catalyzed by nonspecific phosphatases
or perhaps the specific FDPase. In the analysis of inter-
mediates (Table h.h) traces of FlP were observed during the
metabolism of glucose in DD31 and in for cells induced with
fructose or sucrose but not glucose. However the signifi-
cance (in terms of the analytical procedure) of FlP levels
below about 0.1 mM is not known. The level of FlP re-
quired for endogenous induction may be too low to detect,
or may require more time to accumulate, than was provided
with the experimental procedure used. (In this procedure
increased time of exposure to substrate results in in-
creased dilution of the final extract and hence a loss of
sensitivity in the assays.) The greater degree of induc-
tion of the lowKm PEP system in DD31 and In by fructose

and sucrose than by glucose, as indicated by the Km factor
index values (Table h.3), suggests that the level of FlP

is somewhat lower in the mutants growing on glucose than

29h

that in the wild type during growth on fructose. The higher
specific activity of the PEP system in DD31 and 1h than in
the'wild type may be due to an abnormally high degree of
induction of the Km factor causing increased activation.
There are some interrelated anomalies in accumulation
patterns and acid production rates in Table hxh. First,
for DD31 and even more so for 1h, the FDP levels are
abnormally low in unexpected circumstances. The somewhat
lower levels of FDP in the presence of only glucose were
reproducible for fructose-and glucose-grown cells of DD31.
Glucose-grown cells of DD31 and 1h should have normal
FDP levels in the presence of glucose. That they do not:
suggests that FlPK has a role of some sort in constitutive
metabolism. One such role of course'would be the avoidance
of endogenous induction of the PEP systemywhich, once it
occurs, may cause minor side effects. The lack of constitu-
tive fructose metabolism associated with loss of FlPK, as
indicated by loss of acid production from fructose in
glucose-grown cells of DD31 and 1h, is also evidence for
a constitutive role for FlPK. This is discussed further in
Section h.2.S. Second, mutant 1h had only a h to 7-fold
increase in FlP in the presence of only fructose. Addition
of glucose resulted in a much higher level of FlP. Since
there is apparently no way for mutant 1h to metabolize
fructose, accumulation may be limited by the availability

of PEP. Addition of glucose would provide a source of PEP.

 

295

Third, in the presence of only fructose DD31 and 1h had
low'to negligible levels of FDP as expected since produc-
tion of FDP from fructose should occur slowly or not at

all in these mutants. However addition of glucose did not
produce normal FDP levels, suggesting that the production
of FDP from glucose might be inhibited by the accumulation
of FlP. The relatively normal levels of G6? suggests that
phosphoglucose isomerase [which is inhibited by FlP in
mammals (119),and in A, aerogenes,‘V. Sapico, unpublished
dati] is functioning normally. The absence of accumula-
tion of F6P suggests that F6PK is also unaffected by the
accumulation of FlP. (Another possibility is that either
GoP or F6P is a feedback inhibitor of the source of GéP

so that accumulation of these two intermediates is sup-
pressed.) The almost complete absence of FDP in 1h in

the presence of both glucose and fructose could be attributed
to the cyclic PEP phosphatase system mentioned above which
might irreversibly withdraw PEP at a high enough rate to
cause a decrease in the steady state levels of the inter-
mediates in glycolysis. Note that DD31 had a normal rate
of acid production from glucose in the presence of fructose.
This suggests that the inhibition of growth on glucose by
FlP does not occur at the level of carbohydrate degradation
in DD31. However for mutant 1h grown on fructose, acid
production from glucose‘was inhibited by the presence of

fructose.

 

 

 

 

 

 

 

296

Although it is not critical for an explanation of this
series of mutants, it has not been possible yet to establish
definitely that all of the fructose is excreted during the
growth of in RAF on sucrose since some experiments of N. E.
Kelker suggest that it is possible that at least some of
the intracellular fructose produced from sucrose is con-
verted to FlP‘without leaving the cell. At the abnormally
high level of fructose'which is probably present, phospho-
transferases other than the PEP system such as the H-phos-
photransferase (Section 2) may function in the formation
of FlP. Another speculation is that fructose approaching
an induced enzyme II unit from the inside surface of the
membrane encounters a binding site with a high Km, whereas
that approaching from the other side encounters a Km factor--
adapted low'Km site. If the PEP system is at all reactive
toward intracellular fructose it appears that it must have
a higher Km for intracellular than for extracellular fruc-
tose. This is because the growth of DD31 at a normal rate
on sucrose indicates that a toxic accumulation of FlP does
not occur. This is also indicated by the relatively low
level of FlP which accumulates during growth of DD31 on
sucrose as compared with the level present in in (Table
h.h). The lock of accumulation implies that the constitu-
tive low'Km PEP system in DD31 (which has a Km for fruc-
tose-which is in the same range, 2 x 10"“L M, or much lower

than that of the 6-FK is not able to phosphorylate, at a

297

maximal rate, the normal low level of intracellular fructose
produced from sucrose, whereas the 6-FK is. The low level
of FlP accumulation in DD31 during growth on sucrose (Table
h.h) may be due to the presence of a small amount of fruc-
tose (0.05%) in the sucrose used in the experiment. How-
ever if this accumulation of FlP in DD31 does result from
the phosphorylation of intracellular fructose by the PEP
system it would represent a rate of no more than about 15%
of that which the PEP system is capable of catalyzing as
indicated by comparison with the level to which FlP
accumulates in 1h during the utilization of sucrose (Table
h.h). Thus if the PEP system in 1h RAF does phosphorylate
intracellular fructose at a maximal rate it would probably
do so because the fructose level is abnormally high during
the utilization of sucrose. The significance of this line
of reasoning is that it suggests that the PEP system, in

vivo, does not exhibit the same kinetic parameters with

 

respect to extracellular substrate as would be expected of

a carrier mediated transport mechanism. It also suggests
that the results, discussed in Section 3.3.h, obtained with
an in 21312 system by Kaback (101), may also be true for an
in 3123 PEP system. An inability of PEP systems to phospho~
rylate intracellular substrate would not be particularly
surprising. One would not expect to find cytoplasmic ATP-
dependent kinases involved in the phosphorylation of sugars
produced intracellularly by the hydrolysis of disaccharides,

as exemplified by sucrose metabolism in A. aerogenes, if

298

PEP systems could perform this function. It seems quite
possible that some of the "missing kinases" (86) might be
found if cells were grown on the appropriate di-, tri-
and polysaccharides.

As mentioned in Section 1.1,Liss, Horwits and Kaplan
(7) have predicted that fructose is metabolized by an FlP

pathway of some sort in A, aerogenes. It was proposed that

 

mannitol—l-phosphate was the actual inducer of mannitol-l-
phosphate dehydrogenase and that the constitutive level of
the enzyme was proportional to the intracellular concentra—
tion of F6P (which is convertible to mannitol—l-phosphate

by this enzyme). If this were the case then the constitutive
level of mannitol-l-phosphate dehydrogenase in cells grown
on a particular substrate should be proportional to the
intracellular level of F6P produced during the metabolism

of that substrate. Of eight substrates tested fructose
failed to give the results predicted on the basis of known
or assumed pathways (via F6P in the case of fructose).
Therefore, (instead of dismissing the theory) they suggested
that fructose was metabolized via FlP rather than F6P.

Our results of course confirm this. Unfortunately the F6P
levels in the experiments given in Table h.h were too low

to be determined reliably. However the G6P levels might

be used as a rough index of the-F6P levels. As seen in
Table h.h, in wild type cells fructose did not produce a
detectable level of G6P, indicating probably that fructose

299

is indeed a poor inducer of mannitol-l-phosphate dehydrogenase
because a low level of F6P is present during metabolism via
the PIP pathway. The low level of G6P is also expected on
the basis of the analysis of an FDPase-negative mutant as
presented in Section h.2.6.

We summarize the results of the analysis of mutant
DD31 and its derivatives as follows. First, the rudimentary
model for fructose metabolism (Figure h.3) is supported.
However this model is probably an oversimplified description
even for wild type cells and must be substantially modified
and extended in order to explain the metabolism of fructose
by mutants. It is definitely indicated that FlPK is a
step in the metabolism of exogenously supplied fructose.
It is definitely indicated that 6-FK is responsible for

the phosphorylation of fructose in the metabolism of sucrose.

It is indirectly indicated that the PEP system for fructose
does not interact in the same way with intracellular sub-
strate as with extracellular substrate. It is indirectly
indicated that FlPK functions constitutively in the avoidance
of the endogenous induction of an operon which responds to
FlP and which controls the induction of at least the Km
factor and possibly FlPK. It is proposed that free intra-
cellular fructose is the inducer of operons controlling

the induction of 6-FK and sucrose. It is proposed that the
metabolism of fructose via two different pathways is a
consequence of the transport of exogenously supplied fruc-

tose by a PEP system.

 

 

300

h.2.5. CONSTITUTIVE METABOLISM OF FRUCTOSE. As seen from
the acid production rates given in Table h.h glucose-grown
'wild type cells metabolize fructose at a low rate. In
Figure h.h is shown the time course for similar assays of
acid production. In the presence of mineral medium-2

(the standard mineral medium, Section l.h.l, minus magne-
sium sulfate and ferrous sulfate) glucose-grown cells exhibit
low initial rates which increase within 10 to 20 minutes,
apparently due to induction of FlPK and the Km factor.

In another experiment it was found that under normal
growth conditions FlPK was fully induced in cells pre-
viously grown on mannitol within 15 minutes of exposure

to fructose. As seen in Figure h.u, if the mineral medium
-2 is replaced by 0.85% NaCl induction does not occur
(even for incubations of twice the length of that shown

in Figure h.h) and the rate on glucose is only 20-25% of
that in the presence of mineral medium-2. As little as
0.1 ml of mineral medium-2 per 5 ml incubation mixture
containing 0.85% NaCl was enough to cause normal induction
and the normal high rate of acid production. Cells could
be washed in either 0.85% NaCl or mineral medium-2 with-
out effecting the results of the assays (provided the
cells used were relatively free of contaminating mineral
medium-2). These results are no doubt due to the require-

ments of enzymes in catabolic pathways for various ions

301

 

 

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303

and perhaps more important, the requirement of certain ions
for transport or the association of ion transport with
sugar transport (11h). The saturation curves in Figure
h.5 for acid production from fructose indicate that the
constitutive component of fructose metabolism is rather
minor.

In some initial experiments on constitutive metabolism
an assay for acid production containing only NaCl was used.
The results obtained were inconclusive because, as it now
appears, a lack of the proper ionic conditions causes
severe defects in metabolism which may include not only an
overall inhibition, but also changes in the pathways
involved,due to mechanisms similar to those observed in
mutants lacking FlPK or 6-FK. Thus it is not at present
certain what pathway is responsible for constitutive metabo-
lism. The results described in Section h.2.6 suggest that
the pathway does not involve production of F6P, isomeriza-
tion of fructose to produce glucose, reduction of fructose
to produce sorbitol or mannitol,or any other pathway.
resulting in a product which is convertible to F6P or 06?.
It can be seen in Table h.h that loss of constitutive acid
production from fructose was associated with loss of FlPK
even though this enzyme cannot usually be detected in
crude extracts of glucose-grown cells (the low value in

Table 1.2 is an exception). FlPK has however been detected

 

 

 

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306

in certain fractions during the purification of F6PK from
glucose-grown cells (ll, 12) so that it may be that the
enzyme is simply present at too low a level to be detected
easily in crude extracts of uninduced cells. There is also
some tentative evidence that enzyme 1 is required for the
constitutive metabolism of fructose although the investiga-
tion of enzyme I - negative mutants is still in a preliminary
stage. Hopefully, further investigation will establish a

definite relationship between the transport step in the con- F

 

stitutive metabolism of fructose and the constitutive high

Km PEP system for fructose.

307

h.2.6. PLEIOTROPIC MUTANTS LACKING FDPase OR F6PK. It
became evident in the course of isolating mutants that
most mutants with impaired metabolism of fructose also
exhibited impaired metabolism of other carbohydrates,
sometimes in quite incomprehensible patterns. To examine
the growth patterns exhibited by pleiotropic mutants an
isolation was made during the latter stage of the isola-
tion work in which cells were expressed on a variety of
substrates so as to randomize the selective pressure

to a certain extent. Mutagenized cells were expressed
1,000-fold on glucose, mannose, citrate, nutrient broth
and glycerol. They‘were then treated with penicillin in
the presence of fructose or glucose and plated on fruc—
tose-EMB agar. Any colony which varied even slightly from
the wild type was picked. The results of patch testing ab-
normal colonies on agar containing various substrates are
given in Table h.5. Some of the nutrient broth-positive,
carbohydrate-negative mutants are probably due to lesions
in biosynthetic pathways supplying components present in
nutrient broth. However the majority of these mutants
were capable of slow growth on one or more of the carbohy-
drates tested. If this group of 50 mutants is scored, not
relative to the wild type as was done, but with respect to
the growth on the carbohydrate on which the mutant grew
best, then about 27 different growth patterns are found.

Nutrient broth-negative mutants generally exhibited slow

 

Table h.§.

See text for procedures.

308

MUTANT GROWTH PATTERNS FOR A RANDOM ISOLATION.

 

 

 

No

(1311
total)

 

+ Nutrient

agar (NA)

Fructose (F)

 

1; :?
+3
A A E
9 a a v
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33‘ 8 ‘3 2'3 Type
O O C‘. C:
{>3 :5 C3. C4
H H (0 (O
- - - - all-
+ + + + all+ (Wild Type)
+ _ - - NA+ Gy-+ ("aldolase-"l
+ + + + F-
_ + + + F" Gy’("FDPase_")
+ _ _ _ F+ Gy+ ("FOPK-I'")
- + - - 6+
— + + + Gy-
+ + + + NA-
_ + ++ - cy‘ Mtl"
+ + - - NA- M- Mtl—
+ - + + F” G-
+ - + + G-
— + - + F- Gy- M-
- - +~ - NA+ M+

 

 

 

309

growth on all of the substrates without the variations shown

by many of the nutrient broth-positive mutants. Most of

 

these mutants probably have lesions in the biosynthesis and
functioning of complex molecules which limit growth to a
slow rate that is independent of the nonlimiting metabolism
of carbohydrates.

Two common types of mutants found (Table h.5) were
those negative only on fructose and glycerol and those
positive only on fructose and glycerol. If fructos§ is

metabolized primarily via FlP its metabolism like that of

 

glycerol would not require the participation of F6PK,
whereas that of glucose, mannose and mannitol which produce
F6P would require this enzyme. Thus the fructose-, glycerol—
positive phenotype could be due to a loss of F6PK. Further-
more, if the 6-FK induced by fructose were completely non-
functional in fructose metabolism then fructose would be a
completely gluconeogenic substrate, like glycerol, and
would require the participation of FDPase in its metabolism
in order to provide F6? for biosynthesis. Thus a loss of
FDPase would produce the fructose-negative, glycerol-
negative phenotype. unfortunately most of the mutants
isolated in these groups were not saved. Hawever, a mutant
with the FDPase phenotype, 012, had been isolated pre-
viously. Its growth curves are given in Figure h.l.K. .A
mutant,A9-l, which grew at about 25% of the rate of the

‘wild type on glucose, mannose, and mannitol but normally

310

on fructose and glycerol was isolated by R. L. Anderson (92)
by means of replica plating. Both 012 and A9-l were char-
acterized by V; L. Sapico and R. W. Walter (ll, 92). Mutant
A9-1 was found to lack F6PK. .As noted below'FéPK mutants
have been isolated recently in g..ggli. However prior to
this investigation, the only F6PK-negative mutants known
‘were those found in man (120, 121). Mutant 012 was found

to have lost that component of the FDPase activity
representing the gluconeogenic enzyme. Other mutants

such as 08 and 010 (Figuresusl Gland H) had normal FDPase

 

activities. 012 retains a minor component of FDPase
activity‘which is observed at low pH values and which is
believed to be due to a nonspecific acid phosphatase (92).
These results are consistent in all respects'with'the
proposed pathway for fructose metabolism via FlP. The
analysis of 012 provides the most conclusive evidence
that 6-FK is nonfunctional in the metabolism of exogenously
supplied fructose and‘was a factor in suggesting that un-
phosphorylated intracellular fructose does not occur to
an appreciable extent during fructose metabolism due to
transport via a PEP system.

A similar study of FDPase has been made in g. 9211
and has recently expanded to give results allowing a com-
parison of fructose metabolism in g. 221; and g, aerogenes.

Gotto and Pogell (69) in 1962 found in g. coli a sugar

311

phosphate phosphohydrolase with a pH optimum in the acidic
range which was active with hexose monophosphates as well

as FDP. It was thought that this enzyme was the gluco-
neogenic FDPase because it was induced to much higher

levels by growth on glycerol, acetate and other gluconeo-
genic substrates. Somewhat later Fraenkel and Horecker (80)
isolated a mutant of g. 2213 lacking growth on glycerol

and other gluconeogenic substrates but not fructose and
glycolytic substrates. This mutant lacked the true
gluconeogenic FDPase which occurred at a level about one-
tenth as high as that of the acid phosphatase found by Gotta
and Pogell (69). The enzyme was not removed by osmotic
shock as was the acid phosphatase and showed no variation

in level between cells grown on gluconeogenic and glycoly-
tic substrates (80). In A, aerogenes the FDPase lacking

in 012 occurs at a much higher level than the activity at
low pH, but as in g, ggli_it appears to be present at

about the same level in cells grown on either gluconeo-
genic or glycolytic substrates (92). The ability of the
VFDPase-negative mutant of §°.EEL£ to grow on fructose

was expected since it was thought that fructose was metabo-
lized via F6? in E'.22ll' This hypothesis was based on the
analysis of a mutant, MM6, which was thought to lack a "hexo-
kinase" which presumably,formed F6? and which was for

some reason difficult to assay 01,53). Recently it was found

that MM6 is an enzyme 1 mutant (103). Subsequently, Fraenkel

 

 

 

 

312

has established that, as in.A, aerogenes, there is an
inducible PEP system for fructose in.§. 921; which produces
F1? rather than F6? and that there is also an inducible
FIPK (22, 113). Using a rationale similar to-that used in
the isolation of mutant A9-l, Morrissey and Fraenkel (122)
have isolated three F6PK-negative mutants. As determined
by colony diameter on agar one of them grows well on fruc-
tose, one does not, and one is variable. These results

are not interpretable insofar as evaluation of the FlP
pathway in g. Sflll is concerned. However Fraenkel has
found that a mutant of g. 221$ lacking phosphoglucose
isomerase and glucose-6-phosphate dehydrogenase does not
grow'on fructose under certain conditions in which FDPase
is thought to be inhibited due to toxicity effects,although
this is not definitely indicated by the data (22). The
growthton fructose exhibited by several FDPase mutants
which were isolated previously may be due to another path-
‘way producing F6P from fructose or other complexities (22).
Since the enzyme 11 activity for fructose found by Fraenkel
is inducible, it is not clear at present exactly how it is
related to the constitutive enzyme II for fructose in g.
ggli_examined by Kundig and Roseman (101) (see Section
3.3.3),or if both systems produce the same product. ‘The

results obtained with g. coli and A, aerogenes complement

each other in many respects. Our results substantiating

 

 

 

313

the in vivo significance of_the FlP pathway in A, aerogenes,
suggest that it is also of significance in g. coli even
though this has not been directly demonstrated due to the

difficulties mentioned above. .At the same time the problems

 

encountered by Fraenkel and his collaborators in the analysis
of a larger number of F6PK and FDPase mutants than we have
examined,suggest that this area of metabolism may be more
complex than is apparent from our results. This possibility

is considered further in the next section.

 

31h

h.2.7. A MUTANT LACKING ALDOLASE ACTIVITY. As described
in Section h.2.6 differences have been encountered be-
tween E. 2211 and A, aerogenes in the mutational analysis
of fructose metabolism, one interpretation of which is
that metabolism in the immediate area of F6PK and FDPase
is more complex than is presently apparent. This section
presents the preliminary analysis of a mutant lacking L—
aldolase activity. In this case the results are similar
in almost every respect with a previous analysis of an

aldolase mutant of E. coli and both investigations suggest F

 

that gluconeogenesis involves the participation of a
presently unrecognized pathway.

Mutant u8 (Figure h.l.L) which was obtained in the
randomized isolation (Table h.5) grows only on certain
gluconeogenic substrates not including fructose. The same
phenotype was found in an g. 2211 mutant, h8, which was
characterized as lacking aldolase by Bock and Neidhardt
(123, 12h). .As seen in Table h.1 mutant h8 lacks only
aldolase of the enzymes tested. 0f the substrates tried,
mutant h8 did not grow on glucose, fructose or gluconate
but did grow’on glycerol, succinate and nutrient broth.
The phenotype of mutant u8 is identical in those respects
compared. In addition it does not grow'on pentoses. It
is inhibited to only a moderate extent by glucose. Growth
of mutant h8 was completely inhibited by very low levels
of glucose and it was established that glucose is first

 

 

 

315

 

converted to gluconate, which is excreted and which continues
to inhibit growth as it is slowly metabolized. The inhibi-
tion was attributed to a 7 to 20 fold accumulation of FDP.

Mutant h8 is a temperature sensitive mutant which was isolated.

 

under conditions which probably would have detected only a
glucose toxic mutant (the indicator agar was nutrient broth
plus glucose). As suggested by Bock and Niedhardt (12h)

an aldolase-negative mutant should not grow on glycerol since

the aldolase reaction is the only reaction known to be of

 

metabolic significance in the gluconeogenic conversion of
triose to hexose and pentose. For mutant h8 it was suggested
(12h) that either there may be an alternative route from the
three carbon level to the five and six carbon level, perhaps
a "biosynthetic" aldolase, or that perhaps the aldolase in
mutant h8 was leaky enough to suffice for gluconeogenesis,
although no aldolase activity could be detected even in

cells grown at low’temperature. The phenotype of mutant h8 .
is such that it cannot be easily attributed to the presence

of a leaky aldolase. hB grows almost completely normally

on glycerol and other gluconeogenic substrates. This

suggests that gluconeogenesis proceeds relatively normally

in h8 without the perturbation that might be expected if

metabolism were dependent on a leaky enzyme. A substantial

fraction of the carbon flow in for instance, glycerol metabo-

lism, must be in the gluconeogenic direction and if the

aldolase were leaky enough to permit this, then h8 should

 

316

exhibit a slow’rate of growth on glucose. However h8 grows
at an extremely low rate on glucose, giving no detectable
growth in 13 hours and requiring 2h to h8 hours for
appreciable growth (growth after 2h hours may be due to
reversion). Since glucose appears to be relatively non-
toxic it is difficult to rationalize the lack of utiliza-
tion of a leaky aldolase in glucose metabolism as being

due to toxicity, as might be the case for mutant h8.

Thus it appears that gluconeogenesis in h8 is not
explained by trivial effects. However there is a definite
possibility that the mutant as presently characterized may
not be as simple genetically as assumed (see note below).
As suggested by Bock and Neidhardt, a biosynthetic aldolase
reaction of some sort would fit with all of the available
evidence. Examination of the aldolase(s) of A, aerogenes
might indicate that the glucogenic enzyme does not
catalyze condensation under 13_31!g conditions. Some
aldolases exhibit substantial variations in cleavage vs
condensation rates, depending on the assay conditions (see
reference 125 for an example). The mammalian aldolase B,
as mentioned previously (Section 1.3.h), has properties
(low'Km values for triose phosphates, a relatively high
Vmax for condensation) suggesting special adaption for a
role in gluconeogenesis (37). Rather than producing FDP
a special biosynthetic aldolase might catalyze the formation

of sedoheptulose-l,7-diphosphate to produce F6P by the

317

pathway suggested by Horecker and others (126) in which a
phosphatase and transaldolase convert the sedoheptulose-l,
7-diphosphate to F6P. If the FDPase of A, aerogenes also
catalyzed the hydrolysis of sedoheptulose-l,7-diphosphate

as does that in mammalian liver (12?) then the existence

of this pathway would not contradict the results of the
analysis of 012. The mammalian FDPase is an exception;

all other specific FDPases examined so far, including that
of g, ggll_(128), do not hydrolyze sedoheptulose-l,7-diphos-
phate (129). However for FDPases, including that from g.

coli, purification problems and unusual effects on activi-

 

ties appear to be fairly common, suggesting that 13 vitro
data may not be conclusive. Another possibility is that
if a suitable three carbon ketol donor were available 1g

vivo, such as hydroxypyruvate (130, 131), then from glyceral-

 

dehyde-B-phosphate, transketolase could catalyze the forma-
tion of sedoheptulose-7-ph03phate which would be converted
to fructose-6-phosphate by the nonoxidative enzymes of

the hexose monophosphate pathway. .At least one reaction in
this pathway would have to be essentially irreversible in
order to explain the phenotype of mutant h8. Transfer of

a ketol group from hydroxypyruvate should be, unlike most

transketolase reactions, irreversible, since C02 is a pro-

duct in this case (131). If such a pathway were the only
one available for gluconeogenesis this would contradict

the results of the 012 analysis. However there may be two

 

 

 

 

318

different pathways producing F6P, the utilization of which
depends on metabolic control factors. It has been found
that there are in g, 3211 two different ways in which the
enzymes of the hexose monophosphate pathway are used for
biosynthetic purposes. According to Sable and his colla-
borators (116) (see also references 132-13h) during glucose
metabolism the favored route is the oxidative pathway which
consists of pentose phosphate production starting with 06?
oxidation by glucose-é-phosphate dehydrogenase. During
acetate metabolism the favored route is the nonoxidative
pathway‘which starts‘with condensation of F6? and glyceral-
dehyde-B-phosphate in a transketolase reaction and ultimately
results in pentose phosphates. It is equally possible that
different gluconeogenic substrates follow different routes
to F6P. The differences in the growth of glucose-grown and
nutrient broth-grown wild type cells on glycerol and pentoses
(Figures h.l.A and h.1.A.2) might in some way be related to
inducible gluconeogenic enzymes. As mentioned, the specific
FDPases in g. 2211 and A. aerogenes are not inducible. How-
ever in E. 231$ the phosphatase of Gotto and Pogel (69) is
inducible, suggesting that such an enzyme might be involved
in an alternative gluconeogenic pathway. The increased
utilization of the nonoxidative pathway during acetate
metabolism in g. 2211 found by Sabel et al (116) was also
apparently due to an induced change rather than a rapid

change at the kinetic level.

 

 

319

As supported by examination of labeling patterns in fer-
mentation products, in A. aerogenes and g. 9211, pentose
metabolism occurs via the nonoxidative enzymes of the hexose
monophosphate pathway to produce F6P and glyceraldehyde-3-
phosphate (126, 135). In the cyclic version of the hexose
monophosphase pathway proposed for mammalian tissues (136)
the F6? is recycled so that the only product of the pathway
is glycera1dehyde-3-phosphate. The complete lack of growth
of h8 on pentoses supports the idea that the F6P is metabo-
lized via aldolase and suggests that cycling does not occur,
even to a relatively minor extent. Since pentose toxicity
has not been tested it is possible that F6P might be irre~
versibly trapped as FDP in the F6PK reaction during pentose
metabolism so as to inhibit growth on pentoses in which case
metabolism via the cycle would not be observed. However the
relative lack of toxicity of glucose suggests this is not the
case and also suggests that during exposure to glucose, as
well as pentoses, recycling of the hexose monophosphate
pathway does not occur. It is interesting to note that of
the three F6PK-negative mutants isolated by Morrissey and
Fraenkel (122) as mentioned above, the one which grew on
fructose normally did not grow on D-xylose or L-arabinose.
The other two mutants were not tested on these substrates.

The possibilities given above and others could be
examined by analysis of derivatives of AB lacking growth

on gluconeogenic substrates and their revertants. However

320

such a procedure is likely to become very complex due to the
number of reactions involved in the hexose monophosphate
pathway. It would first be necessary to establish by isola-
tion of other similar mutants that hB is a typical mutant
of its type and that it is easily revertible. [In one
experiment h8 was forced to grow'on glucose (accidentally)
over a period of days and the resulting cells contained a
normal level of aldolase;) In particular it is necessary
to establish that it is not possible to isolate aldolase-
negative mutants which do not grow on gluconeogenic substrates
as'well as glycolytic substrates. If such a mutant exists
then h8 would probably be a revertant of it, so that the
gluconeogenic bypass of aldolase in h8 would not be found
in the wild type. Actually for a rigorous exclusion of
this possibility it‘would be necessary to transfer the
aldolase lesion in h8 to the wild type by recombination.
This is presently impossible since there is no recombina-
tion technique available for A, aerogenes.

[Notez Lack of a technique for recombination is the
most serious obstacle in the analysis of mutants of A,

aerogenes. This is basically the reason why mutational

 

analysis of this organism has been employed primarily as

a means of providing support for _i_r_1_ 3.1.22.9. results rather
than as a means for initiating completely new lines of
investigation. ‘Without a recombination technique allowing
transfer of lesions back to the wild type we have found it

321

risky to invest the time required to find the lesion in a
mutant with a growth phenotype which has no apparent rela-
tionship to existing in vitro results or to general theory.
The main problem in this type of situation is that a mutant
may be a pseudo-revertant of another mutant or in some other
way more complex genetically than is apparent. Reverte-
bility to an apparent wild type is not a particularly good
indicator that the mutant is not a pseudo-revertant of some
type. For instance, if MB were a pseudo-revertant as
described above it is easily conceivable that it might be
revertible to an apparent wild type. Mutant 1h RAF (Section
h.2.h) is essentially a pseudo-wild type strain. In fact,

A. aerogenes provides the outstanding example of the ability

 

of bacteria to rapidly acquire new metabolic pathways by
genetic modifications (83). A recombination technique, since
it would allow one to place as much confidence in the signifi-
cance of mutant growth phenotypes as in other types of data,
'would allow mutational analysis to lead, rather than follow,
an $2,!it£g_investigation. In the present case the signifi-
cance of mutant h8 is enhanced considerably by the existence
of mutant h8 in E, 22£1_for which the lesion is transferable
to the wild type (123). However h8 is a good example of the
problems that have arisen, when in the absence of the genetic
analysis afforded by a recombination technique, mutational

analysis was extended beyond a rather rudimentary stage.

 

 

322

n.3. METHODS '

h.3.l. MUTANT ISOLATION. See Table h.6 for the particular
conditions used in the isolation of each mutant. Ten m1
overnight cultures of A, aerogenes, PRL-R3, grown on glucose
‘were harvested by centrifugation and suspended in 5 ml of
mineral medium containing 0.2 M ethyl methane sulfonate
(Eastman) (137). After incubation at 32°C for 2 hours with
agitation the cells'were harvested, washed once with 10 m1
of mineral medium, and suspended in mineral mediumscontaining
0.5% Substrate as indicated (Table h.6) and allowed to grow
so as to give a fold increase in cells as indicated. The
cells were harvested,'washed once in mineral medium, sus-
pended in 10 m1 of mineral medium containing 0.5% of the
indicated substrate at the indicated cell density. The
culture was shaken at 32°C until the O‘D'600 doubled, or

in the case of low cell densities, until the 0.0.600 of

a higher density inoculum doubled. Penicillin G (Calbio-
chem) was added at a concentration of 2,000 units/ml and

the culture was incubated at 32°C for the indicated time.
Samples were taken from the point at which phase contrast
microscopy' indicated that about 90% of the cells had lysed
and was continued for no more than 3 hours after this time.
Lysis, as indicated by the first appearance of partial spero?
plasts, usually began about 90 minutes after penicillin was

added. About 200 cells per plate were plated on either

 

323

 

 

 

 

 

o mmoo.o + a mm.o : mos x m ooaooson on ooaosso mHo
miniomopoaem m 00H x m onoosfio oooH Hopoohfio m:
a mmoo.o + a mm.o : mos x a ooaoosae oood ooaosao p00
a mmoo.o + m mm.o a mom a m.H oaoooson on oooosaa mo
a mmoo.o + a mm.o m.m mos x m.” ooooosan on oooosso oHo
o.mmoo.o + a mm.o : mos x H ooooosan oooa ooaacoa. mmooo
o mmoo.o + n mm.o : mos x a ooaoosom oood oaaosao ”mom
a mmoo.o + n mm.o m was x m oaoooaon 000“ m z _xoommmm
mEMisnooosnm espouse you 000“ “ouoohao mfimm
o.mmoo.o + a mm.o m was x m ooaoosau ooos Hoaopaao naoo
Aaoaoaasv . -
some on Ha\oamoo ooooaonsm anon oomaoonsm poops:
aoooomaaH ,aaansosaon coauuaaoxm.

 

 

 

 

.ZOHH¢AOmH HZ<HD§ mOm wmmDQmoomm

.o.s omoae

 

32h

mineral-agar (1.5% agar in mineral medium) containing 0.5%
fructose plus 0.005% glucose or fructose-EMB agar (Difco,
1.0% fructose) as indicated and generally the smaller or
paler colonies were picked and transferred to nutrient agar
and allowed to grow up to about half of the maximum size
possible. The nutrient agar plates were either replica
plated to glucose-, fructose-, mannose-, mannitol- and
glycerol-mineral agar and nutrient agar or else colonies
were suspended in 1 m1 of mineral medium and applied in
patches to fructose- and glucose-mineral agar and nutrient
agar. If replica plating was used the desired colonies
were retested as patches on agar.

In the initial growth testing of mutants every attempt
was made to keep the generations of growth between the
first detection on agar and the first growth curves in
liquid medium as low as possible in order to avoid rever-
sion. Some mutants were isolated which reverted at such
a high frequency that it was not possible to characterize
and maintain them. Mutants were stored in lyophilized form
at -20°C. Before use in experiments lyophilized cells were
'repurified. In repurifying mutants it was found that the
best procedure was*to select 5-10 colonies from agar and

to test each one for growth in liquid medium thus avoiding
the possibility of selecting a slightly modified revertant.

Fructose-EMB agar was particularly good for detecting

heterogeneity in a mutant strain.

 

 

325

14.3.2. ANALYSIS OF INTERMEDIATE LEVELS IN MUTANTS (TABLE Ll.Ll).
To a 500 ml overnight culture of glucose-grown cells 500 ml

of fresh medium was added, cultures were incubated for 30
minutes, 0.25% of the inducing substrate was added and cul-
tures were shaken for h hours at 32°C. Cells were harvested
by centrifugation at 0°C, washed in cold mineral medium-2
(standard mineral medium minus magnesium sulfate and ferrous
sulfate) and suspended in mineral medium-2 (3.0 ml/l.0 g wet
ad). [In one experiment it was found that cells of mutant

1h RAF induced with sucrose contained a high level (about

 

10 mM) of Flewhen incubated (by the procedure described be-
low) in the absence of added sugar. In this particular case
the cells had been tested immediately (10 min) after har-
vesting. The accumulated FlP was completely eliminated by
washing the induced cells twice, resuspending them in mineral
medium (6 g‘wet‘wt/lOOO ml) in the absence of substrate and
incubating for 2 hours with shaking at 32°C. The starved
cells were then harvested andtreated as described above;]

On a Sargent pH Stat, 5.0 m1 of cell suspension in a 10 ml
beaker containing a small magnetic stirring bar (stirrer

set at 10)'was equilibrated at 32°C, neutralized to pH 7.0
‘with dilute HCl and titration‘was initiated with 0.1 M NaOH.

The NaOH was standardized against 0.100 M potassium acid
phthalate. Incubation with stirring was allowed for 8
minutes (endogenous rates were zero after 2 to 3 minutes)

and hOO pmoles of either fructose or glucose, uoo pmoles of

 

 

326

each, or 200 pmoles of sucrose was added and timing was

initiated at that point at which acid production began, if

it began within one minute of the addition of sugar. If

acid production was delayed or did not occur, timing was
initiated one minute after addition of the sugar.. In some
cases, after a 5 minute exposure to hOO pmoles of fructose,
hOO‘pmoles of glucose were added and incubation was continued

for another 5 minutes. The incubations were terminated by

 

addition of 0.2 m1 of 70% HClOu; the suspension was stirred

 

for 5 minutes on a magnetic stirrer; and the beakers were

 

placed on ice for 15 to 30 minutes. The incubation mixture
was neutralized with 1.00 ml of KHC03 (all g/100 ml of solu-
tion, 1.0 m1 of which resulted in a final supernatant pH

of 7.3-7.6) added in several aliquots with stirring to remove
dissolved C02. Suspensions were centrifuged, and the super-
natants were stored at ~2OOC. For assays the supernatants
were unfrozen, checked to make sure the pH was 7.5, and the
residual precipitate of potassium perchlorate'was allowed to
settle. Assays for FlP and FDP were made as described in
Section 3.h.3. For assays of G6P and F6P the assays contained
in a volume of 0.15 ml, 20 pmoles glycylglycine, pH 7.5, 0.1
pmole TPN, and 0.07 ml of supernatant. The reaction was
initiated with the addition of glucose-6-phosphate dehydro-
genase and, once G6P had been utilized, phosphoglucose
isomerase was added to determine F6P. The O‘D'3h0 was moni~

tored continuously. The results were expressed relative to

 

 

 

 

327

the total cell volume which was determined to be 90% (in mi)
of the wet weight (in grams) by exclusion of bovine serum
albumin (138). The‘wet weight contained in the cell sus-
pension used was determined by the formula: volume of

suspension (m1) x 0.D.6 x 6.20 = wet weight (mg).

00

u.3.3. DETERMINATION OF ACID PRODUCTION. The procedure was
the same as described in Section h.3.2 except that 0.01 M
NaOH was used and the incubation mixture contained (1.0 ml
of mineral medium-2, h.0 ml of H20 and 0.05-0.2 ml of a
suSpension of cells in mineral medium—2 (1 g wet wt plus

3 m1 mineral medium-2). In the absence of substrate no

acid was produced. 3 moles of acid were produced per mole

of hexose utilized.

h.3.h. ISOLATION OF.A BACTERIOPHAGE. A bacteriophage was
isolated accidentally when lysis occurred during growth of

a ho liter culture of A, aerogenes, PRL-R3, on 0.5% fruc-

 

tose in a 50 liter carboy under sterile conditions except
that unfiltered air, direct from the air lines, was'used
for vigorous sparging. The inoculum consisted of 5 liters
of overnight cultures of cells grown in Fernbach flasks
under standard conditions (Section l.h.l). In several
months previous to the lysis of this culture five other
cultures had been grown under the same conditions without
evidence of lysis. However two years previously lysis

occurred for unknown reasons in a sterile 100 liter culture

 

328

grown in a Fermicell fermentor. For about six months after
lysis of the no liter culture cells could not be grown under
the same conditions without lysis (subsequently this became
possible). The phage gave clear plaques of 1-2 mm diameter

 

'with cells of A, aerogenes grown mineral agar containing 0.5%
carbohydrate. It has been observed that for an infected
culture in liquid medium (initial O'D‘6OO = 0.2-0.3), subse-
quent to an initial lysis which occurs in about one genera-
tion of growth (see reference 139 for an example), growth
°continues and reaches a normal maximum.O.D.600 after about

6 hours. The phage was found not to infect a number of

 

”strains of E. 2211, Pseudomonas several other bacteria
(particularly those which might be common contaminants in
this laboratory) (lho). Although it is not_known to be of
any significance, the initial culture‘which lysed was about
two feet from several flasks containing a substantial amount
of 1531. This could conceivably indicate a mutation to
virulence of a lysogenic phage. It has been noted that
occasionally, apparently at random, EMS treated cells fail
to grow except after a very prolonged incubation. (Such
cells were not used in the isolation of the mutants

described in this work.)

 

 

 

h.

11.

12.

13.

1h.

15.

REFERENCES

Kamel, M.'Y., and Anderson, R. L., Bacteriol. Proc.,
82 (1965).

Hanson, T. E., and Anderson, R. L., 3. Biol. Chem.,

2Q} 16AM (1966).
Crane, R. K., The Enzymes, 6 56 (1961).

 

 

Fraenkel, D. 0., Falcoz-Kelly, F., and Horecker, B.
L., Proc. Natl. Acad. S33. g.§., 52 1207 (l96h).

 

Fraenkel, D. 0., and Horecker, B. L., J. Bacteriol.,
29 837 (1965).

Kamel, M. Y}, and Anderson, R. L., Arch. Biochem.
Biophys., 1gp 322 (1967).

Liss, M., Horwitz, S. B., and Kaplan, N, 0., J.
Biol. Chem., 237 l3h2 (1962).

Hanson, T. E., and Anderson, R. L., Egg. Proc., 25
S25 (1966).

Rutter, W. J., The Enzymes, 5 353 (1961).

 

Lowry, O. H., and Passonneu, J. V}, 3. Biol. Chem.,
gag 910 (1969).

Sapico, V. L., Ph.D. dissertation, Michigan State
University, 1969.

 

Sapico,‘V. L., and Anderson, R; L., g. Biol. Chem.,
in press (1969). '

 

 

Kelker, N. E., Hanson, T. E., and Anderson, R. L.,
Bact. Proc., 1&3 (1969).

Lowry, O. H., Rosebrough, N. J., Farr, A. L., and
Randal, R. J., A. Biol. Chem., 193 265 (1951).

Slein, M. W., Cori, G. T., and Cori, C. F., g. Biol.
Chem., 186 763 (1950).

 

329

 

19.

20.
21.

22.
23.
2h.
25.
26.

27.
28.
29.

30.

31.

32.

33.

3h-

330

Richards, 0. C., and Rutter, W. J., A. Biol, Chem., 236
3185 (1961).

McRorie, R. A., and Novelli, G. D., Nature 182 150h
(1958). ‘

 

 

McRorie, R. A., Philbrook, G. E., and Payne, W. J., Egg.
Proc.,1§ 287 (1959).

Hollman, 8., Ngg-Glycolytic Pathways of Metabolism g:
Glucose, Academic Press, New York (196h).

Ashwell, G., Methods Enzymol., 5 190 (1962).

Reeves, R. E., Warren, L. G., and Hsu, D. 8., A. Biol.
Chem., 221 1257 (1966).

Fraenkel, D. G., J. Biol. Chem., 223 6h58 (1968).

 

Williamson, J. R., in Chance, B., Estabrook, R. W.,
and Williamson, J. R., (Editors), Control g: Energy
Metabolism, Academic Press, New York, 1965, p. 333.

 

Hsu, D. 8., and Reeves, R. E., 232. Proc., 25 525 (1966).

Leuthardt, F., Revista Espanola gg Fisiolo ia, 16 (supp
1) 107 (1960).

Renold, A. E., and Thorn, G. W., Am. A. Mgg., 19 169
(1955).

Cori, C. F., Biol. 8 osia, 5 131 (19h1).
Cori, G. T., and Slein, M. W., Fed. Proc., 6 2h5 (l9h7).

Leuthardt, F., and Testa, E., Helv. Physio . Acta,§g 67
(1950).

Hers, H. C., Le Metabolisme QB Fructose, Editions Arscia,
Brussels, 1957.

Cori, G. T., Ochoa, 8., Slein, M. W., and Cori, C. F.,
Biochim. Biophys. Acta, 1 30h (1951).

Hers, H. C., and Kusaka, T., Biochim. Biophy . Acta, ll
M27 (1953).

Leuthardt, F., Testa, E., and Wolf, H. P., Helv. Chem.
Acta, 36 227 (1953)-

Kranhold, J. F., Loh, D., and Morris, Jr., R. C., Science,
{25 ho2 (1969).

 

.1111!!! (NIH...

 

331

35. Rutter, W. J., Blostein, R. E., Woodfin, B. M., and
weber, C. 8., Adv. 265. Reg., 6‘39 (1963).

36. Peanasky, R. T., and Lardy, H..A., g. Biol. Chem., 233
365 (1958).

37. Morse, D. E., and Horecker, B. L., Adv. Enzymol., 31
125 (1968).

38. Gomori, G., 3, Biol. Chem., 1L8 139 (19h3).

 

39. Pontremoli, 8., Luppis, B., WOod, W, A., Traniello, 8.,
and Horecker, B. L., ;, Biol. Chem., 2L9 3h6h (1965).

no. Hers, H. G., 3, Biol. Chem., 21;_373 (1955).

hl. Scrutton, M, C., and Utter, M. F., Ann. Rev. Biochem.,
31 2&9 (1968).

h2. Grossbard, L., and Schimke, R. T., 3. Biol. Chem., 221
35h6 (1966).

h3. Mackler, B., and Guest, G. M., Proc. Soc. Exptl. Biol.
Mfiflo, 23 327 (1953).

hh. Lardy, H., The Enzymes,_6_68 (1962).

 

 

 

 

h5. Leuthardt, F., and Wolf, H. P., Clinica Teraputica,
2§_(no. 6) 519 (l96h).

h6. Penhoet, E., Rajkumar, T., and Rutter, W. J., Proc.
Natl. Acad. Sci. g,§., 56 1275 (1966).

h7. Bacon, S. D., and Bell, D. J., Biochem. 6,,‘A2_397 (19h8).

 

 

h8. Fiskeé C. H., and SubbaRow, Y., A, Biol. Chem., 66 375
(192-).

h9. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.,
Manometric Techniques, Burgess, Minneapolis, 1957,
p. 272.

50. Roe, J. H., 3, Biol. Chem., 69] 15 (l93h).
51. Morris, D. L., Science, 19] 25h (19MB).

52. Warburg, 0., and Christian, W., Biochem._Z_., 310 3811
(l9h2)-

53. Khym, J. X., and Cohn, W, E., 6, Am, Chem. Soc., 25f1153
(1953).

 

 

 

 

511..

55.
56.

S7.
58.
S9.

60.
61.

62.
63.
6h.
65.
66.
67.
68.
69.

70.
71.

332

Khym, J. K., Doherty, D. G., and Cohn, W. E., 6, Am,
Chem. Soc., 16 5523 (195A).

Hara, Y., J62. 6, Biochem., A6 571 (1959).

Travelyan, W. E., Proctor, D. P., and Harrison, J. A.,
Nature, 166 huh (1950).

Dische, 2., and Devi, A., Biochim. Biophy . Acta, 32
1110 (1959).

Schroeder, W., and Hockseman, H., 6, Am. Chem. Soc.,
81 1767 (1959).

Peterson, B. A., and Sober, H. A., Methods Enzymol.,
_S_3 (1962).

Levin, 0., Methods Enzymol., 5_27 (1962).

 

 

 

Dixon, M., and Webb, E. C., Enzymes, Academic Press,
New York, 196k p. 152.

Boyer, P. D., Lardy, H., and Myraback, K., Thg Enz es,
5

Rogers, D., and Reithel, F. J., Arch. Biochem. Biophys.,
8‘2 97 (1960). —‘ _'

Dvorak, H. F., Brockman, R. W., and Heppel, L. A.,
Biochemistry, 6 17h3 (1967).

Lee, Y., and Sowokinos, J. R., A, Biol. Chem., 2A2
226h (1967).

Hafkenscheid, J. C. M., Biochim. Biophys. Acta, 161
582 (1968).

Von Hofstein, B., and Porath, J., Biochim. Biophys.
Acta, 64 1 (1962).

Nossal, N. G., and Heppel, L. A., A, Biol. Chem., 22;
3055 (1966). ' ““ _

Gotto, A. M., and Pogell, B. M., Biochem. Biophys. Res.
Comm., 2 381 (1962).

Torriani, A.-M., Biochim. Biophys. Acta, 333 h60 (1960).

 

Stevens-Clark, J. R., Conklin, K. A., Fugimoto, A., and
Smith, R. A., A. Biol. Chem., 223 hh7h (1968).

 

'12.
73.
7h.
75.
76.
77.
78.
79.
...
81.
82.
83.

811.
85.

86.

87.

88.

89.

333

StevenseClark, J. R., Theisen, M. C., Conklin K. A.,
and Smith, R., 6, Biol. Chem., 243 hu68 (1966)

Smith, R. W., and Theisen, M. C., Methods Enzymol., 9
h03 (1966).

Fujimoto, A., and Smith, R. A., Biochim. Biophy . Agtg,
56 u91 (1962).

Holzer, M., Burrow, D. J., and Smith, R. A., Biochim.
Biophys. Acta, _5_6 1.91 (1962). __

Fujimoto, A., ingram, P., and Smith, R. A., Biochim.
Biophy . Acta, 96 91 (1965).

Kundig, W., Kundig, F. D., Anderson, B., and Roseman,
s.,.g. Biol. Chem., 291 3203 (1966).

Schaefler, 8., and Schenkein, 1., Proc. Natl. Acad.
§21o !5§-. 62 1210 (1969).

Theisen, M. C.” Ph.D. dissertation, University of
California, 1965.

Fraenkel, D. G., and Horecker, B. L., J, Bacteriol.,
29 837 (1965).

Englesberg, E., watson, J. A., and Hoffee, P. A.,
Cold Springs Harb. 62m. Ought. 5121-, 26 261 (1961).

Kundig, W., Gosh, 8., and Roseman, 3., Proc. Natl. Acad.
Sci. U;§.,352 1067 (196h).

Anderson, R. L. 'and Wood, W. A., Ann. Rev. Microbiol.,
in press (1969).

Pardee, A. B., Science, 162 632 (1968).

Tanaka, 8., Lerner, S. A. and Lin, B. C. C., Q.
Bacteriol., 93 6h2 (1967).

Wood, W. A., App. Bgy., 35 521 (1966).

Tanaka, 8., and Lin, E. C. C., Proc. Natl. Acad. Sci.
9.6., 5] 913 (1967)."

Egan, J. B., and Morse, M. L., Biochim. Biophyg. Acta,
91 310 (1965). “““"

Egan, J. B., and Morse, M. L., Biochim. Biophy;. Acta,
199 172 (1965).

 

 

90.

91.

92.

93.

9h.

96.

97.

98.

99.

100.
101.
102.
103.

10h.

105.

106.

107.

3311
Egan, J. B. and Morse, M. L., Biochim. Biophys. Acta,
112 58 (1966)

Wang, R. J., and Morse, M. L., 6, M01. Biol., 32 59
(1968).

Sapico, V. L., Hanson, T. E., Walter, R. W., and Anderson,
R. L., J. Bacteriol., 96 51 (1968).

 

KundigS W., and Roseman, 8., Methods Enzymol., 2 396
(1966 .

Hanson, T. E., and Anderson, R. L., Egg. Proc., 21 6hh
(1968)-

Hanson, T. E., and Anderson, R. L., Proc. Natl. Acad.
66;. U.§., 6; 269 (1968).

Morse, M. L., Hill, K. L., Egan J. B., and Hengstenberg,
W., 2. Bacterial. ,95 2270 (1968).

Lin, E. C. C., Levin, A. P., and Magasanik, B., 3. Biol.
Chem., 235 192h (1960).

 

 

Simoni, R. D., Smith, M. F., and Roseman, 8., Biochim.
Biophys. Res. Comm., 3; 80h (1968).

Brew, K., Vanaman, T. C., and Hill, R. L., Proc. Natl.
Acad. §21- g.8., 59 h9l (1968).

Kaback, H. R., J, Biol. Chem., 223 3711 (1968).
Kundig, W., and Roseman, 8., Fed. Proc.,2§ h63 (1969).
Kepes, A., Biochim. Biophys. Acta, 49 70 (1960).

Tanaka, S., Fraenkel, and Lin, E. C. C., Biochem.

Biophys. 26;. Comm., 21_63 (1967).

Levinthal, M., and Simoni, R. D., A. Bacteriol., 91
250 (1969).

Simoni, R. D., Levinthal, M., Kundig, F. D., Kundig,
W., Anderson, B., Hartman, P. E. and Roseman, 8.,
Proc. Natl. Acad. Sci. 6.6., 58 1963 (1967).

Hengstenberg, W., Penberthy, W. K., and Morse, M. L.,
Egg. Proc., 21 6h3 (1968).

Kennedy, E. P. , and Scarborough, G. A., Proc. Natl.
£922. §21 2- §. 5. 225 (1967)

108.

109.

110.

111.

112.
113.
11h.
115.
116.
117.

118.
119.

120.

121.

122.

123.

12h”

 

 

335

Hengstenberg, W., B an, J. B., and Morse, M. L., 3.
Biol. Chem., 263 1 81 (1968).

Hengstenberg, W., Egan, J. B., and Morse, M. L., Proc.
Natl. Acad. Sci. 936., 58 27h (1967).

 

Hoffee, P., Englesberg, E., and Lamy, F., Biochim.
Biophy . Acta, 79 337 (196h).

Mitchel, P., in Kleinzeller, A., and Kotyk, A.,
(Editors), Membrane Transport and Metabolism,
Academic Press, New‘York (1960), p. 22.

Kamel, M. Y., Ph.D. dissertation, Michigan State
University, 1965.

Fraenkel, D. G., J. Biol. Chem., 2y} 6u51 (1968).

Hempfling, W. P., Hofer, M., Harris, E. J., and g
Pressman, B. C., Biochim. Biophys. Acta, 22; 391
(1967). A i

Mortlock, R. P., Fossitt, D. D., and Wood, W. A.,
Proc. Natl. Acad. Sci. U.S., 5g 572 (1965).

Yaphe, W., Christensen, D. D., Biaglow, J. E., Jackson,
M..A.. and Sable, H. 2., Can. ;. Biochem., 22 91 (1966).

 

Wu, H. C. P., and Kalckar, H. M., Proc. Natl. Acad.
S21. g.§., 55 622 (1966).

Wu. H. c. P., g. 121. Biol., 22 213 (1967).

Zalitis, J., and Oliver, 1. T., Biochem. J., 102
753 (1967).

Tarui, 8., Okuno, G., Ikura, Y., Tanaka, T., Suda, M.,
and Nishikawa, M., Biochem. Biophys. Res. Comm., ;9
517 (1967). “‘*“' - “'“'

Layzer, R. B., Rowiand, L. P., and Ranney, H. M.,

 

9 Arch. Neurol., l] 512 (1967).

Morrissey,.A. T. E., and Fraenkel D. G., Biochem.
Biophys. Res. Comm., 32 h67 (1968)

 

Bock, A., and Neidhardt, F. C., J. Bacteriol., 22
h6h (1966).

Bock, A., and Neidhardt, F. C., J. Bacteriol., 92
A70 (1966).

 

 

125.

126.

127.

128.

129.

130.

131.

132.

133.

13h.

135.

136.

137.

138.

139.

lh0.

 

336

Fish, D. C., Ph.D. dissertation, University of
Michigan, 196R.

Horecker, B. L., Pentose Metabolism Lg Bacteria,
John Wiley and Sons, New York, 1962, p. 16.

8., Traniello, 8., Luppis, B., and
3. Biol. Chem., 220 3h59 (1965).

Fraenkel, D. G., Pontremoli,
B. L., Arch. Biochem. Biophys.,

Pontremoli,
Wood, W..A.,

 

8., and Horecker,

L111 14(1966).

Rosen, 0. M., Rosen, S. M., and Horecker, B. L.,
Arch. Biochem. Biophys., 112 hll (1965).

 

 

Racker, E., de la Haba, G., and Leder, I. G.,_2.

Am. Chem. Soc., Z5 1010 (1953).

 

de la Haba, G., Leder, . G., and Racker, E.,

Q_. Biol. Chem. ,_2_Q h09 (1955).

R., and Rittenberg, D., Proc. Natl.

Caprioli,
g.§., 69 1379 (1968).

Acad. Sci.

 

Nat .

and Rittenberg, D., Proc.

Caprioli, R.,
g. 6. , 61 1u22 (1968).

Acad. Sci.

Caprioli, R., and Rittenberg, D., Biochem., 6.
3375 (1969).

Wood, W. .A., in Gunsalus, I. C., and Stanier, R.
Y., (Editors), The Bacteria, Academic Press,
New'York, (1961):'V. 2, p. 59

Katz, J., and WOod, H. G., 3. Biol. Chem., 235
2165 (1960).

Loveless, A., and Howarth, 8., Nature, 18h 1780
(1959).

Kotyk,.A., and Hofer, M., Biochim, Biophys.
102 hlo (1965).

Acta,

Ph.D. dissertation, Michigan State

Mayo, J. W.,
1968.

university,

Ph.D.
1969.

Dahms, A. S., dissertation, Michigan State

University,

 

 

 

 

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