V— - 'u'w wax—3mg.“

 

4-:-
‘—

' ' I ' :3, ----- AEROBACTER AEROGENESE 1; ;.: i

PROPERTIES AND FUNCTIONS 0F
THREE BACTERIALKINASES
PART! AHEXOKINASE SPECIFIC FOR ’f ‘ '
D; MANNOSEANDD‘FRUCTOSEFROMsIV s ~ ,
LEUcou’osmc MESENTEROIDES _  ‘: *

 

, PPP: "'

6 PHOSPHOFRUCTOKINASE FROM

 
  
   

 

Them for the Dir-ii: :7- if
MICHIGAN STATE:

  
  

LIBRARY

Michigan 33” ‘
Universxty

  
     

This is to certify that the

thesis entitled
Properties and Functions of Three Bacterial Kinases
Part I: A Hexokinase Specific for D—Mannose and
.D-Fructose from Leuconostoc mesenteroides
Part II: l-Phosphofructokinase and 6-Phosphofructo—
kinase from Aerobacter aerogenes
presented by

 

Virginia L. Sapico

has been accepted towards fulfillment

 

of the requirements for

Ph.D. degree in Biochemistry

R. L. mm

Major professor
Date June 26 1 6

0-169

 

 

 

 

 

ABSTRACT

PROPERTIES AND FUNCTIONS OF THREE BACTERIAL KINASES
PART I: A HEXOKINASE SPECIFIC FOR D—MANNOSE AND
D—FRUCTOSE FROM LEUCONOSTOC MESENTEROIDES
PART II: 1-PHOSPHOFRUCTOKINASE AND 6—PHOSPHOFRUCTO-
KINASE FROM AEROBACTER AEROGENES
By

Virginia L. Sapico

Part I describes a hexokinase (adenosine 5'—trin
phOSphate:hexose 6-phOSphotransferase) specific for D-
mannose and D—fructose. The enZyme was purified to

apparent homogeneity from extracts of Leuconostoc

 

mesenteroides. D—Mannose and D-fructose were phOSphory—

 

lated at equal rates, whereas D-glucose and 29 other
sugars and sugar derivatives tested were not phosphorya
lated and did not inhibit the enzyme. The apparent Km
value for either hexoSe or adenosine 5'-triph08phate
(ATP) varied with pH, but was independent of the concen_
tration of the other. D—Mannose was a competitive
inhibitor of D-fructose. Product inhibition occurred
With adenosine 5'-diphOSphate (ADP) (competitive with
ATP) but not with D-fructose 6-phOSphate. The pH-act1v_

ity curves were different for the two hexoses, with the

 

D-mannokinase to D—fructokinase ratios being about 1.0

at pH 6.9, 0.5 at pH 8.5. and 0.3 at pH 8.9. The enzyme

 

 

Virginia L. Sapico
had a molecular weight estimated at 47,000. The products
of the phosphorylation of D-mannose and D—fructose were
identified as D-mannose 6—phOSphate and D-fructose 6-
phOSphate, reSpectively. The basis for the unusual
Specificity of the enzyme can be rationalized from an
inSpection of molecular models of the preferred conformau
tions of u-D-mannopyranose and BgD-fructofuranose.
Although one of the models consists of a six—membered
ring and the other a five—membered ring, the positions
of equivalent atoms on the two models are superimposable.
Such topological similarity is not mimicked by D-glucose
or any of the other sugars tested as possible substrates.

Part II describes the functions, properties, and

control mechanisms of 1—phOSphofructokinase and 6»phOs—

 

phofructokinase from Aerobacter aerogenes. Analysis of
mutants lacking 6uphosphofructokinase and fructose
1,6-diphosphatase indicated that Dmfructose metabolism
in this organism is primarily through Dwfructose l—phos_
phate rather than Dafructose 6~phosphate.
6—PhOSphofructokinase was purified sixnfold from
extracts of A. aerogenes PRL~RB. 1~PhOSphofructokinase
was purified 315—fold from extracts of a 6-phOSphOfructO=
kinaseless mutant. Comparative studies on the two enzymes
indicated that they are governed by different control
mechanisms. 6-Phosphofructokinase exhibited a sigmoidal
dependence of rate on D—fructoseoémP concentration,

Whereas 1-phOSphofructokinase exhibited hyperbolic

Virginia L. Sapico
dependence of rate on D-fructose-l-P concentration. ATP
inhibited both enzymes under conditions of Mg++ to ATP
ratios below 2:1. Inhibition of 6-phoSphofructokinase
by.ATP was relieved by Mg++, D-fructose-6AP. ADP, and
various other nucleoside diphOSphates. In contrast,
only Mg++ was found to relieve inhibition of 1-ph03pho-
fructokinase activity by ATP. Both enzymes showed a
sigmoidal dependence of rate on Mg++ concentration.
Increased levels of Defructose-é-P shifted the 6-phos-
phofructokinase curve from sigmoidal to hyperbolic,
whereas D—fructose-l-P had no effect on a similar plot
for i-phosphofructokinase. Other nucleoside triphos-
phates were used as phoSphoryl donors by both enzymes,
and inhibited activity under conditions of Mg++ to
nucleotide ratios below 2:1. In contrast with the
result with.ATP, the inhibition of 6-ph08phofructokinase
by other nucleoside triphOSphates could not be relieved
by D-fructose-6-P. Citrate, D-fructose 1,6-diphosphate,
and D-fructose-ééP inhibited the 1-ph03phofructokinase
reaction competitively with D-fructose-iéP, suggesting
possible $£;E222 control of activity. The data indicated
that whereas 6-ph08phofructokinase exhibits allosteric
properties and a regulatory pattern typical of 6-phos-
phofructokinases from a variety of organisms, 1-ph08pho-
fructokinase behaves more like a non-allosteric kinase.
The molecular weight of 6-ph08phofructokinase was esti-
mated as 100,000, and that of i-phOSphofructokinase as

 

 

Virginia L. Sapico
75.000. The apparent KIn of l—phOSphofructokinase for
either substrate did not vary with the concentration of
the other. This finding is consistent with a sequential

mechanism of substrate binding to the enzyme.

 

 

 

PROPERTIES AND FUNCTIONS OF THREE BACTERIAL KINASES

PART I: A HEXOKINASE SPECIFIC FOR D-MANNOSE AND
D—FRUCTOSE FROM LEUCONOSTOC MESENTEROIDES

PART II: l-PHOSPHOFRUCTOKINASE AND 6—PHOSPHOFBUCTO-
KINASE FROM AEROBACTER AEROGENES

By

Virginia L. Sapico

A.THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

 

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1969

 

 

ACKNOWLEDGMENTS

The author wishes to eXpress her gratitude to
Dr. Richard L. Anderson for his patient guidance and
encouragement throughout the course of this work.
Thanks are also due to T. E. Hanson for providing
some of the mutants used in these studies, to B. W.
Walter for doing some of the enzyme assays described
in Part II, Section A, and to all fellow researchers
for their helpful suggestions. The help of Mrs.
Shirley Randall in the preparation of this manuscript

is also appreciated.

ii

 

TABLE OF CONTENTS

 

Page
ACKZNOWLEDGMEIVTS o o B o o O D 0 o o 9 o o 0 O 9 0 ii
LIST OF TABLES I) D 9 O o o a o O o o c o 0 o o 0 Vi

LIST OF FIGURES 0 O D O O 0 O D O O 0 O I O 0 P 0 vii

LIST OF ABBREVIATIONS . . . o . . o . . . . . o 0 X1

PART I. A HEXOKINASE SPECIFIC FOR D-MANNOSE AND
D-FRUCTOSE FROM LEUCONOSTOC MESENTER-

OIDES 0 0 O 9 O 0 D 0 O O 0 9 G O O 0 O

H

Introduction . . . . . .
Materials and Methods . . . . . . . .
Source and confirmation of identity
of Leuconostoc mesenteroides
Growth of cells and preparation of
extracts . . . . . . . . . . .
Chemicals . . . . . . .
Mannofructokinase assays . .

0 O 0 0 0 ° 0

O 0

O

9 0

 

Other enZyme assays . . .
Protein measurements . .
Disc gel electrophoresis . . . .
Sucrose density gradient centrifu-
sation . . . . . . . . . . . .
Cellulose acetate electrophoresis .
Results . . . . . . . . . . . . . . . .
Phosphorylation of heXoses with ATP
by L. mesenteroides cell
extFacts . . . . . . . . . . . . . 11
Requirements for Dumannose and D-
fructose phosphorylation by cell
extracts stored for 24 hr at
0-20C o o lo a o o o o o o o a 0
Purification of mannofructokinase . . 14

O 9
O 0
D 0 fl
0 o

n
o
0
C
0 d
O

n a u o t a

9 O

HO\O \O\OGDO\(:‘\A) Ki) WH

0
Hi4

IvlnClZ fractionation o a o o o o 114’
First ammonium sulfate fraction»

ation O D o o 0 9 O o 0 9 O o o 14
Heat treatment . . . . . a . . . . 18
Second ammonium sulfate fraction~

ationooooooooooooo 18
Calcium phOSphate gel treatment . 18
DEAE—cellulose chromatography . . is

Sephadex chromatography . . . .

 

iii

PART II.

Page

Further attempts to separate the

two kinase activities . . . . . . . 22
Disc gel electrophoresis . . . . . 22
Cellulose acetate electrohporesis . 23
Sucrose density gradient centri-
fugation . . . . . 24
Thermal inactivation at 60°C . . . 24
Properties of mannofructokinase . . . 2“
Stability . . . . . . . 2h
PhOSphoryl acceptor Specificity . . 24
PhoSphoryl donor Specificity . . . 28
Effect of pH . . . . . . . . . . . 28
Determination of Km for D-mannose
and D- fructose . . . . . . . 33
Determination of Km for ATP . . . . 33

Inhibition of D-fructokinase
activity by D-mannose ..... #2

Product inhibition . . . . . . . 42
Estimation of molecular weight . . #2
Product identification . . . . . . . . 51
DiscuSSIOn o o o o o o o o o o o o o o o 53
summary 0 o o o o o o o o o o o o o o o o 59

1-PHOSPHOFRUCTOKINASE AND 6-PHOSPHO-
FRUCTOKINASE FROM AEROBACTER.AEROGENES . 61

IntrOduotlon o o o o o o o o o o o o o o 61
Section A. Determination of the Rela-
tive Significance of the D-Fructose-
l—P and D-Fructose-6-P Pathways in
A. aero enes by Analysis of Mutants
cEIng 6-PEOSphofructokinase and
D-Fructose 1 ,6-DiphOSphatase . . . . . 64

Materials and methods . . . . . . . . 64
Bacteria O O C O O O O O O O O O O 64
Culture media o_o o o o o o o o o o 64
Growth of cells and preparation

Of eXtraCtS o o o o o o o o o o 65
Chemicals 0 o o o o o o o o o o o o 65
Enzyme assays . . . . . . . . . . . 65
Protein determination . . . . . . . 67

Results 0 O O O O O O O O O 0 I O O 68
Growth pattern . . . . . . . . . . 68
Enzyme activities in cell

' extracts . . . . . . . . . . . . 68

DISCUSSIOn o o o o o o o o o o c o o 0 76

Section B. Purification, Properties,
and Regulation of 6-Ph08phofructo-
kinase and l-PhOSphofructokinase . . . 80

iv

 

 

Review of literature . . . . .
Materials and methods . . . .
Bacteria . . . . . .
Growth of cells and preparat

REFERENCES .

Chemicals . . . . .
EnZyme assays . . 0
Protein determination

Results . . .
Purification of 6-PhOSphof

H0 o o
O
:39 o o

of extracts . .

B
O
O
0

°°‘oo

O’coo

O
O
I
I)
0

ru

t

°O°oo

kinase . . . . .

Protamine sulfate precipita-
tion . .

Ammonium sulfate fractionation

Sephadex G~200 chromatography

Purification of 1-phOSphofructo—

Discussion . . . . . . o . .
Summary of Part II . . . o . .

kinase 0 O 0 (I I 0 O O o 0 O
Protamine sulfate precipita—

tion 0 o o o a o
Ammonium sulfate fractionation
Sephadex G~200 chromatography
Calcium phOSphate gel adsorp-

tion and elution . . .
Ammonium sulfate precipitation
pH fractionation . . .

Properties and regulation of 06.-

phosphofructokinase and 1-
phOSphofructokinase . . . . .
Stability 0 D D O O D 0 O O 0
ATP inhibition . . . a . . . .
Interaction of heXose phOSphateo
substrate with ATP and Mg ++
Effect of Ng++ . . . . . . .
Effect of other nucleoside tri-

phOSphates . . .
Effect of nucleoside diphos—
phate S o O a O 0 o I

Effect of nucleoside mono;
phosphates o o a o o o o o
Substrate Specificity . . . .
Test for inhibition of 6~PFK
by Defructose-lmP .
Inhibition of leFK by
fructosew6~P . . .
FDP inhibition . . 0
Effect Of P1 0 o o 0
Effect of citrate .
Effect of pH . . . .
Molecular weight determ n

0 O 9 O

D
1

0090900.
(1‘

OOH-0.999
0

00500090

0 o o o a a o o 0 o 0 o o o o g

V

0....

O

O I

0 G O O O a O O

Page

80
100
100

100
101
102
103
104

104

104
104
104

106

106
110
110

110
113
113

114
114
114

117
122

 

Table
I.

II.

III.

VI.

VII.

VIII.

X.

LIST OF TABLES

PhOSphorylation of D-glucose, D-mannose,
and D—fructose by cell extracts of
‘L. mesenteroides . . . . . . . . . . . .

Requirements for D-mannose and D-
fructose phOSphorylation inIL.
mesenteroides cell extracts after 24-hr
storage at 0:20C . . . . . . . . . . . .

Purification of mannofructokinase from
‘L. mesenteroides . . . . . . . . . . . .

Thermal inactivation of mannofructo-
kinase at 60°C . . . . . . . . . . . . .

PhOSphoryl donor Specificity of manno-
fructij-nase O O O O O O O O O O O O O 0

Enzyme activities in crude cell
extracts of A, aerogenes . . . . . . . .

Comparison of the properties of

A. aero enes l-PFK and 6-PFK with

Those 0% S-PFKS from other sources . . .
Purification of 6-PFK from A. aerogenes
PRL-3300000000000000000
Purification of l-PFK from mutant A9-1 .

Phosphoryl donor Specificity of 6-PFK
and. 1 -PFK C O O O O C C O O O O O O O 0

vi

Page

12

13

15

27

29

71

81

105
109

130

 

Figure
1.

10.

LIST OF FIGURES
Page

Disc gel electrophoretic patterns of

fractions obtained from the latter

stages of purification of mannofructo-

kinase . . . . . . . . . . . . . . . . . 17

Elution profile of mannofructokinase
on Sephadex G-100 and G~75 . . . . . . . 21

Sedimentation pattern of mannofructo-
kinase and peroxidase standard in a
sucrose density gradient . . . . . . . . 26

pH optima of mannofructokinase . . . . . 31

LineweaverwBurk plots for determining
the Km values of mannofructokinase for
D—mannose and Dufructose . . . . . . . . 35

Lineweaver~Burk plots Showing the rela—
tionship of mannofructokinase reaction
velocity to Dumannose and Dafructose
concentrations in the presence of vary-

ing ATP concentrations at pH 6.9 and

809 D O O D o 0 a G 0 0 0'0 0 0 O O 0 O 37

LineweaveraBurk plots for determining
the Km values of mannofructokinase for
AT? 0 3 0 o 0 0 O O 0 0 0 i 9 0 t 9 o o 39

Lineweaver-Burk plots showing the

relationship of mannofructokinase reac~

tion velocity to ATP concentration in

the presence of varying concentrations

of D-mannose and Dufructose . . . . . . 41

Lineweaver-Burk plot showing the rela_

tionship of mannofructokinase reaction
velocity to D-fructose concentration

in the presence and absence of

D—mannose . . . . . . . . . . . . . . . 44

Kinetic plot for determining the K1 for
D-manno S e o o o o o o o o o o o o o a g 46

 

 

Figure
11.

12.

13.

14.

15.
16.

17.

18.

19.

20,

21.

22.

23,

Lineweaver-Burk plot showing the rela-
tionship of mannofructokinase reaction
velocity to ATP concentration in the
presence of various concentrations of
ADP O O O 0 0 O 0 l 0 O O O O O O O O 0

Kinetic plot for determining the K1
for ADP O ‘9 O O O O D O O 0 O I I O O 0

Structural models showing the topo—
logical similarity between a-D—manno-
pyranose and B-D-fructofuranose . . . .

Growth characteristics of A. aerogenes
PRL—R3 and mutants 012 and A9-1 on
D—glucose, D-fructose, and glycerol . .

pH-activity profile of FDPase . . . . .

Pathways for the metabolism of D-
glucose, Defructose, and glycerol in

A. aerogenes o o o a a o o a a O o 0 o

Elution pattern of 6-PFK on a Sephadex
G-200 column . . . . . . . . . . . . .

Elution pattern of l-PFK on a Sephadex
G-ZOOCOlumn coo-cocooo-oo

Inhibition of 6~PFK by ATP in the
presence of various concentrations of
Mg++ and D—fructose—6-P . . . . . . . .

Inhibition of 1=PFK by ATP in the
presence of various concentrations of
Mg++ and D—fructose-l-P . . . . . . . .

Dependence of initial velocity of 6-PFK
and l-PFK on the hexose phOSphate con-
centration under conditions of varying
Mg++ to ATP ratios . . . . . . . . . .

Lineweaver~Burk plot for determining
the Km of l-PFK for D-fructosemi-P in
the presence of various ATP concentra—
tions 0 O a O O 0 0 O O 0 O 0 0 I I O a

LineweaveraBurk plot for determining
the Km of 1~PFK for ATP in the presence
of various D-fructose—l-P concentra-
tions 0 O O O 9 O O 0 O C O a c O O D .

viii

Page

48

50

55

7O
75

78

108

112

116

119

121

124

126

 

 

 

Figure
24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Page

Dependence of 6-PFK and 1-PFK reaction
velocity on Mg++ concentration in the

presence of various concentrations of

the hexose phosphate substrate . . . . . 128

Dependence of 6-PFK and 1-PFK reaction
velocity on hexose phOSphate concentra-

tion with other nucleoside triphos-

phates as phosphoryl donors . . . . . . 132

Inhibition of 6-PFK by CTP in the
presence of various concentrations of
D-fructose-6-P and Mg++ . . . . . . . . 134

Dependence of 6-PFK reaction velocity
on Defructose-6—P concentration in the
presence of various nucleotides . . . . 137

Lineweaver-Burk plot showing the rela-

tionship of l-PFK reaction velocity to
D-fructose-iAP concentration in the

presence of various concentrations of
D-fmCtose-é-P o o o o o o o o o o o o o 1&2

Kinetic plot for determining the K1 of
1-PFK for D-fructose-6AP . . . . . . . . 144

Lineweaver-Burk plot showing the rela-

tionship of l-PFK reaction velocity to
D-fructose-l-P concentration in the

presence of various concentrations of

FDP. O O O O O O O O O O I O O O O O O O 146

Kinetic plot for determining the K1 of
1-PFKforFDP.............. 148

Lineweaver-Burk plot showing the rela-

tionship of 1-PFK reaction velocity to
D-fructose-i-P concentration in the

presence of various concentrations of
Citrateooooooooooooooooo 151

Kinetic plot for determining the K1 of
1-PFK for Citrate o o o o o o o o o o o 153

pH-activity profiles of 6-PFK and
l-PFKooooooooooooooooo 155

Dependence of the rate of the 6-PFK

reaction on D-fructose-6qP concentra—
tion at different pH values 0 o o o o o 157

ix

Figure
36.

37.

Page
Elution profiles of 1-PFK, 6-PFK, malic
dehydrogenase, alcohol dehydrogenase,
and cytochrome c on a Sephadex G-100
COlmooooooooooooooooo 160

Plot of elution volume Ve vs log MW of
the standards for the estimation of the
molecular weights of 1-PFK and 6-PFK . . 162

 

 

LIST OF ABBREVIATIONS

D-Glucose-6-P, D-glucose 6—phoephate; D-glucose-
l-P, D-glucose 1-phoSphate; D-fructose-64P, D-fructose
6-ph08phate; D-fructose-i-P, D—fructose 1-phOSphate;
Lefructose-i-P, L-fructose 1-phOSphate; D-mannose—6AP,
D-mannose 6-phOSphate; D—mannose-l-P, D—mannose i-phos—
phate; L-ribulose-E-P, L-ribulose 5-phosphate; mannitol-
1—P, mannitol l-phOSphate; FDP, D-fructose 1,6-diphos-

phate; FDPase, D-fructose 1,6-diphosphatase; 1-PFK, D-

 

fructose 1-phOSphate kinase; 6-PFK, D-fructose 6-phos—

phate kinase; Pi. inorganic phOSphate; PPi, inorganic
pyrophOSphate; EDTA, ethylenediaminetetraacetate; triose-

P, triose phOSphate, PEP, phOSphoenolpyruvate; NADP,
nicotinamide adenine dinucleotide phOSphate; NADPH,
reducedfnicotinamide adenine dinucleotide phOSphate; NAD,
nicotinamide adenine dinucleotide; NADH, reduced nico-
tinamide adenine dinucleotide; NTP, nucleoside triphos-
phate; NDP, nucleoside diphosphate; NMP, nucleoside mono-
phOSphate; ATP,.adenosine 5'—triph08phate; ATPase,
adenosine 5'-triphOSphatase; ADP, adenosine 5'-diphosphate;
AMP, adenosine 5'-mon0phOSphate; cyclic AMP, adenosine
3‘.51—cyclic monophOSphate; ITP, inosine Si—triphosPhate;
IDP, inosine 5'—diphOSphate; IMP, inosine 5'-monophosphate;
UTP. uridine 5'-triphosphate; UDP, uridine 5'-dlphOSphate;

xi

 

UMP, uridine 5'-monophOSphate; GTP, guanosine 5'-triphos—
phate; GDP, guanosine 5'-diphosphate; GMP, guanosine 5'-
mcnophosphate; CTP, cytidine 5'-triphosphate; CDP,
cytidine 5'—diphOSphate; CMP, cytidine 5'-monophOSphate;
TTP, thymidine 5'—triphosphate; TDP, thymidine 5'-diphos—

phate.

 

 

 

 

PART I

A HEXOKINASE SPECIFIC FOR D-MANNOSE.AND D—FRUCTOSE
FROM LEUCONOSTOC MESENTEROIDES

INTRODUCTION

Heterofermentative lactic acid bacteria of the
genera Leuconostoc and Lactobacillus ferment hexoses
through a hexose monophOSphate (phOSphoketolase) path-
way (1, 2). Although a constitutive hexokinase (ATP:

hexose phOSphotransferase) has been implicated in initi-

 

ating the pathway (3, 4), other investigators have
reported D-glucokinase activity in extracts to be weak
or undetectable (5, 6). Preliminary investigations in
this laboratory of possible alternative phOSphorylating
mechanisms involving phOSphoryl donors other than ATP
revealed instead high levels of kinase activity for
D-glucose, D-mannose, and D—fructose in fresh extracts of
Leuconostoc mesentergides. Storage of the extracts at
0-2°C for 24 hours, however, invariably caused a dis-
appearance of D-glucokinase activity without affecting
the kinase activity for D-mannose or D—fructose. This
observation substantiated previous indications (6)
that more than one enzyme was involved in the phos-

phorylation of the three hexoses, and suggested that

2
previous failures to detect D-glucokinase activity were
due to the lability of the enzyme rather than to its
absence from the organism. Cifferi, Blakley, and Simpson
(6) have pointed out that it was unknown whether the
apparent phosphorylation of D-mannose and D-fructose in
‘L. mesenteroides was mediated by a single kinase, by two
Specific kinases, or by a kinase Specific for either
D-mannose or Defructose in conjunction with an isomerase
which interconverted the two hexoses. Because D-mannose
isomerase (7, 8) and Specific kinases which phOSphorylate
either D—mannose (9-11) or D-fructose (9-12) at carbon
atom 6 have been found in a variety of organisms, the
latter two possibilities seemed likely. However, evi-
dence presented in this part of the thesis indicates
that the phosphorylation of D-mannose and D-fructose in
‘L. mesenteroides is effected by a single enzyme with a
unique specificity, and that an isomerase which inter-
converts the two hexoses is not involved. These conclu-
sions are based on an investigation of the properties of
the mannofructokinase (ATthexose 6-ph08photransferase
specific for D-mannose and D-fructose) after its purifi-
cation to apparent homogeneity.

The findings described in this part of the thesis
have been published (13). The common identity of the
D-mannokinase and D-fructokinase activities and the
instability of the D-glucokinase activity of'L.;mg§gg-

teroides have recently been confirmed by DeMoss (14).

 

 

MATERIALS AND METHODS

Source and Confirmation of Identity
of_§irfiesenteroid§§

 

The strain of L. mesenteroides (designated LM)
used in this investigation was obtained from Dr. w. A.
Wood. Records denoting the original source of this
strain were not available. Its identity was confirmed
by the following observations: colonies on sucrose agar
plates were large and mucoid, indicating the synthesis
and deposition of dextran around the cells; growth in
glucose broth was accompanied by gas production; and
cells viewed by phase contrast microscopy appeared as
chains of Spheres or short rods.
Growth of Cells and Preparation
pngxtracts

The organism was grown and maintained in LBS
medium (15) as modified by Costilow, Etchells, and
Anderson (16). The medium contained per liter of broth:
10 g of trypticase, 5 g of yeast extract, 6 g of KHZPOM,
2 g of ammonium citrate, 1 g of monosorbitan oleate com-
plex (trade name, Tween 80; purchased from E. H. Sargent
C0,), 20.5 g of anhydrous sodium acetate, 0.6 g of Mgson’
0.1 g of Mnsou, 0.03 g of FeSOu, and 20 g of D-Slucose
(autoclaved separately). The cells were grown without

3

 

 

 

 

4
agitation in 20-liter carboys at 30°C. The inoculum was
1 liter of a 24-hr culture in the same medium. The cells
were harvested with a Sharples centrifuge approximately
30 hr after inoculation and were washed once with dis-
tilled water. The yield was about 2 g (wet weight) of
cells per liter.

Extracts were prepared from washed cell suSpen-
sions in distilled water by treatment for 12 to 15 minutes
in a Raytheon 250-watt, 10—kc sonic oscillator circulated
with ice water. The supernatant obtained after centrifu-
gation of the broken cell suSpension at 16,300 x g was

used as the crude extract.
Chemicals

Horseradish peroxidase (Grade C; A403/A275‘>1.5)
and twice-recrystallized rabbit muscle lactic dehydrogen-
ase (containing pyruvic kinase) were from Worthington
Biochemical Corp., Freehold, N. J. Glucose-6-P isomerase
(A grade, Boehringer) had <0.01% of contaminating activ-
ities of 6—phosphogluconate dehydrogenase, phosphogluco-
mutase, 6-phOSphofructokinase, and glucose reductase,
Glucose-6-P dehydrogenase (Boehringer) contained the fol-
lowing nominal impurities: glucose reductase, <0.05%;
hexokinase, <0.2%; and 6-phosphogluconate dehydrogenase,
<0.01%. Crystalline yeast hexokinase (substantially free
of ATPase, adenylate kinase, glucose~6-P dehydrogenase,

and 6-phOSphogluconate dehydrogenase) was from Sigma

 

 

5

 

Chemical Co.. St. Louis, Mo.

D-Mannose-6-P isomerase was purified from D—glu—
cose-grown Aerobacter aerogenes PRL-R3 (17): An extract
was prepared by treatment of washed cells in 0.05 M
sodium phOSphate buffer (pH 7.0) for 5 to 7 min in a
sonic oscillator, followed by centrifugation of the
resulting broken cell suSpension. Solid (NH4)2804 was
added to the crude extract to a final concentration of
0.1 M, and nucleic acids were precipitated by the slow
addition of 20% by volume of a 2% aqueous solution of
protamine sulfate. Solid (NH4)2804 was added to 50%
saturation to the supernatant from the protamine sul-

fate step. The precipitate was dissolved in 3 ml of

 

 

0.05 M sodium phOSphate buffer (pH 7.0) and the solution
obtained was passed through a Sephadex G-100 column (40

X 2.9 cm) equilibrated with 0.05 M sodium phOSphate buf-
fer (pH 7.0) and eluted with the same buffer. Fractions
of 4 ml each were collected. Fraction 22 was lo-fold

purified over the extract and was free (< 0.1% of D-

 

mannose-6-P isomerase) from 6-phosphogluconate dehydro-
genase, glucose-6-P isomerase, and glucose—6-P dehydro-
genase.

D-Fructose was from Pfanstiehl Lab., Waukegan,
111., Difructose-6-P from Schwarz Bioresearch, D-fructose-
1-P and PEP from Calbiochem, and Damannose-l-P from Sigmao

L—Mannose, L—glucose, L-ribulose, Deallose, D-altrose,

   

 

 

6

D— and L-galactose, and D— and L-xylulose were obtained
as described by Kamel, Allison, and Anderson (17).
L-Fructose was the preparation described by Mayo and
Anderson (18). D—Mannose (c.p. grade) was recrystal—
lized twice (19) to reduce the contaminating D—glucose
to 0.07%, as determined with a stereospecific D—gluco-
kinase (20). D-Mannose-6-P free from D-fructose-6-P and
D—glucose-6-P was prepared enZymically in a reaction
mixture containing 3 mmoles of ATP, 3 mmoles of MgClZ,
3 mmoles of twice-recrystallized Dumannose, and crystal-
line yeast hexokinase. The pH of the mixture was main-
tained at pH 7.5 by titration with 4.1 N NHQOH with the
use of a Sargent recording pH stat. The D-mannose-6-P
formed was isolated as described for L-ribulose-S-P (21),

Pyridine nucleotides and nucleoside di- and tri-
phosphates were from P-L Biochemicals, Milwaukee, Wis,.
The nominal impurities of the latter were as follows:
ITP, <T% IDP; UTP <4% UDP; GTP <4% GDP; ATP, <4% ADP;
GTP, <4% CDP; ADP, <4% each of ATP and AMP; IDP, <4% rm;
and TTP, <4% TDP.

All other chemicals were from standard chemical

sources.

Mannofructokinase Assays

TWO types of assay were employed: a non-Specific

Pyruvate kinase-lactate dehydrogenase—linked assay and a

 

 

 

 

7
Specific glucose—6-P dehydrogenase-linked assay. Both

were done in microcuvettes with a 1—cm light path and
were monitored at 340 nm with a Gilford multiple sample
absorbance-recording Spectrophotometer thermostated at
250C. The units of the coupling enzymes used in the
assays refer to the number of umoles of product formed
per minute at pH 6.9 and 25°C. The pyruvate kinase-
lactate dehydrogenase-linked assay contained in a volume
of 0.15 ml: 8 umoles of glycylglycine buffer (pH 6.9),
0.5 umole of ATP, 1.0 umole of MgClZ, 0.4 uncle of PEP,
0.05 uncle of NADH, 0.26 unit of lactate dehydrogenase,
0.03 unit of pyruvate kinase, 1.0 umole of hexose sub—
strate, and limiting amounts of mannofructokinase. Con—
trols to correct for NADH oxidase and ATPase activities
contained all components of the reaction mixture except
the hex0se substrate. This procedure was used for rou=
tine assays during purification of the enzyme and for
determination of Km values, pH optimum, sugar specificity,
and inhibition by D-fructose-6-P.

The glucosee6~P dehydrogenase-linked assay con_
tained in a volume of 0.15 ml: 8 umoles of glycylglycine
buffer (pH 6.9), 0.5 umole of ATP, 1.0 uncle of MgClz,
0.1 uncle of NADP, 0.08 unit of glucosew6-P dehydrogen—
ase, 0.31 unit of glucose-6=P isomerase, 1.0 uncle of
D-fructose or 1.0 uncle of Dumannose plus 0.005 unit of

mannose—6-P isomerase, and limiting amounts of mannofruci

 

 

 

 

 

 

 

8

tokinase. This assay was used for determining nucleo~
tide specificity, for testing inhibition by compounds
other than D-fructose-6-P, for determining the K1 for
ADP, and for identifying the product.

Identical rates were obtained with both assays.
The rates were linear with time for 12 to 15 min and
were directly proportional to the amount of mannofruc-
tokinase present. A unit was defined as the amount of
enzyme that catalyzed the phOSphorylation of 1 uncle of
D-mannose or D—fructose per minute at pH 6.9 and 25°C.
Specific activity was defined as the number of units

per milligram of protein.
OtherREnzyme.Assays

Peroxidase was determined by measuring 460 nm
absorbance increase of a reaction mixture consisting
of 0.15 ml of 0.003% H202 in 0.01 M sodium phOSphate
buffer (pH 6.0), 2 ul of 1.0% pedianisidine in methanol,
and 1 ul of peroxidase solution of an appropriate dilu-
tion.

Several enzyme activities were measured by adap_
tations of the glucose-6—P dehydrogenaseelinked assay
for mannofructokinase. The assay for 6-phosphogluconate
dehydrogenase contained 1.0 uncle of 6~phosphogluconate
in place of hexose and ATP. Assays for phosphofructo-
mutase and phosphomannomutase contained 1.0 uncle of

Dbfructose-léP or Damannose-luP, reSpectively, in place

 

 

 

 

 

 

9
of D—fructose plus ATP or D—mannose plus ATP. The assay
for adenylate kinase contained ADP in place of ATP.
6-PhOSphofructokinase was measured in the pyruvate
kinase-lactate dehydrogenase~linked assay with D-fructose-

6-P as the substrate.

Protein Measurements

 

Protein was estimated Spectrophotometrically at 280
and 260 nm with the aid of a nomograph based on the data

of Warburg and Christian (22).
Disc Gel Electrophoresis

This was performed in polyacrylamide gels at pH
8.6 according to directions supplied by Canalco (Rockville,
Md.). The lower gel contained 7.5% acrylamide, one-half
the standard amount of N,N,N',N'”tetramethylethylenediamine,
and no K3Fe(CN)6. Ferricyanide was omitted because it
inactivated mannofructokinase. When assays were to be
made on the electrophoresed protein, the lower gel was
sliced lengthwise prior to staining.

Sucrose Density Gradient Centrifugation

This employed a 3—ml linear gradient of 5 to 20%
sucrose in 0.2 M (NH4)ZSO4 (pH 7.0). The gradient was
carefully layered with a mixture of 25 ml (50 ug of pro-
tein) of the concentrated Sephadex Gm75 fraction and 20

01 (10 ug of protein) of peroxidase. The centrifugation

 

 

 

 

 

10
was run for 14 hr at 39,000 rpm in a Spinco model L cen-
trifuge. At the end of the run, the bottom of the tube

was punctured and 5-drop fractions were collected.
Cellulose Acetate Electrophoresis

The cellulose acetate paper strips (2.5 x 12 cm,
trade name: Sepraphore III) were from the Gelman Instru—
ment Co. The barbital-acetate buffer contained 5 g of
sodium barbital and 2.3 g of anhydrous sodium acetate
per liter of solution. The pH of the buffer was adjusted
with HCl to pH 6.0, 7.4, or 8.6. Seven ul (40 ug of pro-
tein) of the kinase preparation were applied on each
paper strip. The runs were conducted at 4°C for 2 hours
with a current of 1 ma per strip. Protein bands were
developed by dipping the strips in 0.2% nigrosine (bac—
teriological stain, Allied Chemical) solution for 1 min,
and immersing in 5% trichloroacetic acid. The proteins
from the unstained strips corresponding to the protein

bands were eluted with 0.2 M (NH4)2504-

 

 

 

 

 

 

RESULTS

~PhOSphorylation of Hexoses With.ATP_

by L. mesenteroides Cell Extracts

AS shown in Table I, fresh extracts of L. m§§gg-
teroides exhibited kinase activity on D—glucose, D-fruc-
tose, and D—mannose at nearly equal rates. Storage of
the crude extracts for 24 hr at 0-2°C, however, resulted
in a complete loss of the D—glucokinase activity but had
no effect on the kinase activity for either D-mannose or
D-fructose. The Duglucokinase activity could not be pro-
tected from inactivation by 4 mM reduced glutathione or
2-mercaptoethanol.

Sonic oscillation of cell suSpensions for varying
time periods (5 to 20 minutes) or freezing of the cells
for 48 hr prior to preparation of the extracts had no
significant effect on the phOSphorylating capacity for

any of the three hexoses.

Requirements for DuMannose and D-Frpgtggg

Phos hor at
p for 2 Hours at O~2 C

Table II shows an agreement of results obtained
from the pyruvate kinase-lactate dehydrogenase-linked
and the glucose—64F dehydrogenase-linked assays for

kinase activity. The specificity for the correSponding

11

 

 

 

 

A|Al

. 4min «lanolpfi.o..lwo.fi ....H| .HO MPOGHPNT HHWAV Ann.
onoposhhln dado .onofiflflfilg .ONOOSHNIAH M0 “01“.”. SA.”

 

H 33“.

 

 

 

12

 

ems
.hcmmm oostHIDmNSomoapmnoo opmpomanomSSaM opmessmm om» mo
an panammmfi macs onHpmamHoflmmosm omopodhmtm one mmonsmzim .hmmmm poMQaHiommmom
nonoSSoo mronmoosHm was no ems an scammmos mos Soapoaasonamosm omoodawim

. . .m.o me see comm so :aopoma
adeHHHHs Hoe Deanna Sea oopmaaaogmmona opmameSm no moaosn “apneapom oamaoo we

 

 

 

 

mw.o no.0 o oomuo‘pm.mhs 3N
.HOH GOHOPm POTHPNG 0659.0 A0.
mm.o mn.o 00.0 Powhpfio DGSHO Smmhm Am
omoposaaim omozsmslm omoousIQ
So wmpaeapom no *apabapow no sandbapom
cacaooam cacaooan oaaaooan.

 

. mooaoeoPSomos .m no mpomapxo HHoo an
omoposamlm one .omonsmsnm .mmooSHwIQ ho Scapeaanosamosm

 

H mqm48

 

 

Complete pyruvate 2
lactate dehydrogem
assay mixture

minus ATP

minus hexose

minus PE”

Complete glucose-6.
dehydrogenase-link:
mixture

minus hexose

minus mimosa-6.
glucose-64° iso
Klucose-6-P de

“111118 whose-6

 

 

13
TABLE II

Requirements for D-mannose and D-fructose phoSphorylation
I [in cell extracts after 24—hr storage at 0-200

 

 

Activity on Activity on

 

 

D-mannose D-fructose
units*/mg unitS*/mg
Complete pyruvate kinase-
lactate dehydrogenase-linked
assay mixture 0.36 0.45
minus.ATPc 0.006 0.006
minus hexose 0.01 0.01
minus PEP 0.006 0.008
Complete glucose-6-P
dehydrogenase-linked assay
mixture 0.36 0.45
minus hexose 0 0
minus mannose-6-P isomerase,
glucose-6AP isomerase, and
glucose-6-P dehydrogenase 0 0
,minus mannose-6-P isomerase 0 0.45

*Units: micromoles of substrate phosphorylated per minute
at 25°C and pH 6090

 

hexose-64’ of t!
dehydrogenase-11
products of D-mz
were D-mannose-t

The lack
mannose-é-P ison
linked assay 1‘01
both mannose isc

crude extracts 11

Pur

-‘

Unless ot
formed at 0-400.
in Table III. D

from the latter
F1101 Fractionat

1511012 (1
eXtract (13 mg p
“0n of 0.05 M.

centrifvsation a
First Ammonium S

Solid amm
ring to the abov

between #0 and 7

mm and d1 sso

 

14

hexose-é-P of the coupling enzymes in the glucoseuémP
dehydrogenaseulinked assay suggested that the principal
products of Damannose and Dafructose phOSphorylation
were D~mannosea6=P and D-fructoseuémP, reSpectively.

The lack of NADP reduction in the absence of
mannose-6—P isomerase in the glucosemomP dehydrogenasem
linked assay for mannokinase indicated the absence of
both mannose isomerase and mannoseuéuP isomerase in the

crude extracts under the assay conditions employedo
Purification of Mannofruotokinase

Unless otherwise stated, all operations were perm
formed at O-DOC. A summary of the purification is given
in Table III. Disc electrophoretic patterns of fractions

from the latter stages of purification are shown in Fig. l.

MnClp Fractionation

MnC12 (l M) was added with stirring to the crude
extract (13 mg protein per ml) to give a final concentram
tion of 0.05 M. The resulting precipitate was removed by

centrifugation and discarded.

First Ammonium Sulfate Fractionation

——-—_—-—-—.—

 

Solid ammonium sulfate was added slowly With stirs

ring to the above fraction° The protein precipitating

between 40 and 70% saturation was collected by centrifum

gation and dissolved in water° This solution contained

 

 

 

 

|C¥OSHHIQV IOpOSHMIQ

14l“¢a‘lfk\ lC¥Cnrlf$ICV
53.5306 hhobooom hpdeducm hpabdpcw
3.30on 330on Hence

 

OOuTOQOOSQH EOHH omdfldflopofihhofifidfi ho fiOdPNOHHHHSN

 

uofldflhfluflommfi.

HHH Wang

 

 

 

 

.m.m mg osm 00mm pm opSQHE Hog oopmfimaosmmosm omoxog mo moHoam

 

 

 

 

unpasbe
oo.H o.wm H.m 0.0m mom m.a . mo: mmuw: mQOHpomae
OOHUU Noumsgwm
oo.H m.dm ma m.:m odaa w.a m: mmumm mmoapooam
omoazaaoonmmmm
oo.H o.Hm mm o.Hm comm 0H.H oma How Nfliomvmmo
, Ho; mam 3:: 3.3 ommm 34 ema : N as was
om 35: 38%
i" 3.0 3: $3 iii” 83. 8.0 3H poem
no; a: we was 3% SJ ems e m gains:
om 3sz 33m.
M No; 3.: mm 3.: 8% No.0 83 macs:
mw.o um.H ooa mw.H oaam no.0 mam: pomapxo Haoo
QHoPOHQ Sampogm
ms\mpass R ms\mpamd empass we
Aommsfix Aommsfix Aommsax AmmoQHM owmauomm¢ maopoaa .Qoapomam
omdfiak0p05hhlm Iondmalmv Iopodsmnmv nonesshnmv lop05HmuQV Hmpoe
ommsfixossm21Q mpflbfipom mambooom mpabfipom mpfibfipom
033on 328mm H309

 

mopHOHomewoa oopmosoodoq Bong ommsHMoposamossma no SoameHwfihsm

HHH mam¢H

 

Figure 1:

16

Disc gel electrophoretic patterns of
fractions obtained from the latter stages
of purification. A, calcium phOSphate
gel supernatant; B, DEAE—cellulose Frac-
tions 36-56; C, Sephadex G-75 Fractions
47 to #9; D, enzyme obtained by eluting
the region correSponding to the darker
band in C. The direction of migration

was down.

Hamel

I.“ d;

ms of

iter stages

_osphate

,ose FraC-

ractions
eluting
darker

gration

Figure 1

 

N I

 

 

 

 

 

 

 

considerable sa

fate in the pre

Heat Treatment

 

The abov<
in an 80°C Hate]

Precipitate was

Second Ammonium
M

Three Vo]
tion (pH 7) were
the above fracti

and dissoIVed in

Calcium Pho hat

Calcium p
added to the abo'
Der m1 of enzyme
and discarded. .
Sephadex c.25 Co:
Phosphate bilffer
eluted With the :

est mannofructokj

D
EAE~Ce11u10 Se C1

The Sephac‘

105 X 23 Cm) of

18
considerable salt because of the occluded ammonium sul-

fate in the precipitated protein.

Heat Treatment

The above fraction was held at 50°C for 3 minutes
in an 80°C water bath and cooled rapidly on ice. The

precipitate was removed by centrifugation and discarded.
Second.Ammonium Sulfate Fractionation

Three volumes of saturated ammonium sulfate solu-
tion (pH 7) were added with stirring to two volumes of
the above fraction. The precipitate was centrifuged down

and dissolved in 20 ml of water.
Qalcium.Phosphate GelETreatment

Calcium phosphate gel (Sigma, 11% solids) was
added to the above fraction at a ratio of 0.04 ml of gel
per m1 of enzyme solution. The gel was centrifuged down
and discarded. The supernatant was passed through a
Sephadex G-25 column equilibrated with 0.05 M sodium
phOSphate buffer (pH 7.0). Fractions (5 ml each) were
eluted with the same buffer, and those that had the high-

est mannofructokinase activity were combined.

DEAE-Cellulose Chromatography

 

The Sephadex fraction was passed through a column

(1.5 x 23 cm) of DEAE-cellulose (Sigma, exchange capacity

 

= 0.9 meq per 1
phate buffer (1
with 225 ml of
fate in a lines
36 to 56, whicl
combined. As a
cant loss in 3;
Disc gel elect:
contaminating F

DEAR Step incre

Se hadex Chrome

The abov
ing With solid ;
centrifuged pre
tion was then I)
SeDhadex 9.100
(pH 7.0). Fl‘ac
0‘2 M ammonium ;
ammonium gulf a t(
Slyoylglycine b1
in enZyme aotivj
a relatively hig
It was not eStat
were actually re

purifiedv 5% rec

ammonium Sulfate

19
= 0.9 meq per g) equilibrated with 0.05 M sodium phos-

phate buffer (pH 7.0). Fractions (3 ml) were eluted
with 225 ml of the same buffer containing ammonium sul-
fate in a linear gradient from 0 to 0.h M. Fractions

36 to 56, which had the highest Specific activity, were
combined. .As shown in Table III, an apparent signifi-
cant loss in Specific activity resulted from this step.
Disc gel electrophoresis (Fig. 1B), however, showed that
contaminating proteins had been removed. Moreover, the

DEAE step increased the.A28O:A260 ratio from 1.1 to 1.6.

Sephadex Chromatography

The above fraction was concentrated by precipitat-
ing with solid ammonium sulfate and dissolving the
centrifuged precipitate in 3.5 m1 of water. This solu-
tion was then passed through a column (2.7 x 38 cm) of
Sephadex G-100 equilibrated with 0.2 M ammonium sulfate
(pH 7.0). Fractions (1.5 ml) were eluted with neutral
0.2 M ammonium sulfate (Fig. 2). Substitution of the
ammonium sulfate as eluent with either water or 0.05 M
glycylglycine buffer (pH 7.0) led to a considerable loss
in enzyme activity, indicating that the enzyme requires
a relatively high ionic strength for maximum stability.
It was not established whether ammonium or sulfate ions
were actually required. Fractions #6 to 55 (50-fold
purified, 5% recovery) were combined, concentrated by

ammonium sulfate precipitation, and passed through a

 

 

 

 

 

 

 

F igure 2:

Top graph, chromatography of DEAE-

cellulose Fractions 36 to 56 on Sephadex

G-100; bottom graph, chromatography of
Sephadex G-100 Fractions 46 to 55 on

e
Sephadex G-75. Details are given in th

text.

 

Figure 2

SEPHA
6400

Z;

 

 

T
l

 

 

(UNITS PER ML,
O

N
O

 

KINASE
E

 

 

 

 

 

(UNITS PER ML)

KINASE

20

3

C)

[9
C)

l5

IO

Figure 2

 

I
SEPHADEX
(3400

  

 

 

 

 

 

 

 

 

   

 

PROTEIN

 

/
KINASE

 

 

 

 

 

 

 

 

 

 

 

 

SEPHADEX

 

 

”(3-75

 

 

 

 

 

 

 

 

 

PROTEIN/-
\)

 

 

 

 

 

 

 

 

 

 

 

20 so 40 so so
FRACTION NUMBER

400

300

200

IOO

C)

400

300

PROTEIN (MICROGRAMS PER ML)

200

IOO

 

 

Sephadex G-50 c
sulfate (pH 'M
activity was at
concentrated, 8
Again, there we
2). Fractions
by ammonium sul
phoresis at thi
one minor prote
The kina

were consistent

1:1 throughout ‘

Further Attem]

Disc Gel Electrc

A sample
(2 mg protein p
disc gel electr-
from Canalco.
pending to the
eluted with 0.2
NO mannofructoki
eluted solutions
moment on the en

the enzyme . Low

K3Fe(CN)6 and. t

 

 

22

Sephadex G-50 column equilibrated with 0.2 M ammonium
sulfate (pH 7.0). No further increase in specific
activity was attained. The peak fractions were combined,
concentrated, and passed through a Sephadex G—75 column.
Again, there was no increase in Specific activity (Fig.
2). Fractions 47 to 49 were combined and concentrated
by ammonium sulfate precipitation. Disc gel electro-
phoresis at this stage showed that the enZyme contained
one minor protein impurity (Fig. 1C).

The kinase activities on Demannose and D—fructose
were consistently present at a ratio of approximately

1:1 throughout the purification described above.
Further Attempts to Separate the Two Kinase Activities
Disc Gel Electrophoresis

A sample (0.1 ml) of the Sephadex G—75 fraction
(2 mg protein per ml) was examined for homogeneity by
disc gel electrophoresis using premixed gel solutions
from Canalco. The portions of the unstained gel corresa
ponding to the protein bands of the stained half were
eluted with 0.2 M ammonium sulfate (pH 7.0) and assayed.
No mannofructokinase activity was detected in any of the
eluted solutions. Tests on the effect of each gel com=
ponent on the enzyme showed that K3Fe(CN)6 inactivated
the enzyme. Lower gel A was therefore prepared without

K3Fe(CN)6 and, to prevent a too rapid polymerization of

 

 

 

 

 

the aerylanide
of the stander
the gel minus
the bands in
from the premi
ing to the dark
D-mannose and E
Homogene
tokinase band (
11m sulfate (pH
a second disc e
the second run
on D-mannose am
activity of the
amount of elute<

determination.
Cellulose Aceta

No sharp
any of the runs
at pH 8.6, mann
only a thin por
The kinase acti

approximately eq

fructokinase act

 

 

23

the acrylamide, the volume of TEMED was reduced to half
of the standard amount. The enzyme remained active in
the gel minus K3Fe(CN)6 and the number and position of
the bands in the gel remained identical to those obtained
from the premixed gel solutions. The section correSponda
ing to the darker band (Fig. 10) exhibited activity on
D-mannose and D—fructose in a 1:1 ratio at pH 6.9.

Homogeneity was attained by eluting the mannofruc=
tokinase band (about 680 ug of protein) with 0.2 M ammon~
ium sulfate (pH 7.0), and using the solution obtained for
a second disc electrophoresis run. The single band from
the second run (Fig. 1D) also exhibited equal activities
on D-mannose and D—fructose at pH 6.9. The Specific
activity of the band could not be ascertained because the
amount of eluted protein was too small for an accurate.‘

determination.
Cellulose Acetate Electrophoresis

No sharp resolution of proteins was obtained in
any of the runs made at pH 8.6, 7.4, and 6.0. However,
at pH 8.6, mannofructokinase activity was eluted from
only a thin portion (about 5 mm) of the protein band.
The kinase activities on Demannose and anructose were
approximately equal in all the slices that had mannoa

fructokinase activity.

 

 

 

    

activity had

Thermal Inactiv

 

Table IV
of the two kina
tivation of D-m

fructokinase ac

25:
Stability
The enzyn

ing the peak fr
stable for over
presence of 0.2

pH 6.9, 8.5, an
Pho hor 1 Aces

0f 32 sug
concentration of

were pho sphoryla

fructokinase in

 

 

 

2L»

Sucrose Density Gradient Centrifugation

Fig. 3 shows the sedimentation pattern of mannon
fructokinase and horseradish peroxidase on a sucrose
density gradient. Each fraction which had D-mannokinase

activity had an equal D—fructokinase activity at pH 6.9.
Thermal Inactivation at 60°C

Table IV shows the rates of thermal inactivation
of the two kinase activities at 60°C. The rate of inac~
tivation of D—mannokinase activity paralleled that of De

fructokinase activity at pH 6.9. 8.5, and 8.9.

Properties of Mannofructokinase

Stability

The enzyme preparation obtained after concentrate
ing the peak fractions from the Sephadex G~75 step was
stable for over 6 months when stored at O=2°C in the
presence of 0.2 M (NH4)2804. The ratios of activity at

pH 6.9, 8.5, and 8.9 remained constant throughout storage.
PhOSphoryl Acceptor Specificity

Of 32 sugars and sugar derivatives tested at a
concentration of 70 mM, only Dumannose and‘Dmfructose
were phOSphorylated. With 0.016 unit of purified manno=

fructokinase in the pyruvate kinasemlactate dehydrogenasea

 

 

 

 

 

 

Figure 3:

25

Sedimentation pattern of mannofructo-
kinase and peroxidase standard (molecu-
lar weight = 40,000) in a sucrose density
gradient. The kinase fractions had equal
activities on D-mannose and D-fructose.

Details are given in the text.

ES
<3

co
0

Figure 3

KINA:

 

 

A
O

 

RELATIVE ACTIVITY
0)
o

20

 

 

 

 

Fl

RELATIVE ACTIVITY

I00

80

60

4o

20

Fig

ure 3

 

_ DéNSITY GRADIENT

 

CENTRIFUGATION

I

 

 

 

'o

 

 

 

 

\.~

KINASE/1

 

-0 _ __.R'PE ROXIDASE_
r ‘- /

 

 

 

 

 

~O

 

i

 

 

 

 

O
_1_ _\

I\

O

 

 

 

 

 

 

-1 \\

‘ 7’

 

 

 

 

 

 

IS 20 24 28
FRACTION NUMBER

(<1: (C(V I In ((1.13... Ctrrfirer+$0C
HQQ ME moov mfikNflm 038 .mhom

¢£¢r>F0H9>HOQ CH UQDGGE mdk Ufld O.m ER on bottlwbd WW3 «HE
omNaémzv Ufiooom 0:9 Swsohnp todhdhsm mGB meQH&ODOSHMOGC$E

I hvd>d#od Odhdoommv Ampm

0000 Dd OWGEHN0903HHOQCNE MO SOHDdeDOGCH HGEHOSH

NVH mama

 

 

2?

 

 

 

 

 

 

 

 

 

 

 

 

o O .0 mm
mm.o m.m mm.o s.m om.O m.m ON
mm.O ma Hm.o ON sm.o ON we
am.o em em.O pm mm.O pm OH
am.O as em.O ms NO.H mm m
Oe.O OOH mm.O NO em.O Hm m
Om.O OOH mm.O OOH O0.0 OOH O
QHE
mmapaeapos mmapaeapom .mmapa>apoe
owmsamoposamnm hpabapom mmmsaxoposawnm hpa>flpoc mmeHMoposaqu apfibapom mEHp
op omeHMoasmanQ HmeHQa op cmmsaxoasmaum Hmapamfi op ommgaxoasmana HmeHQH MQHpmmm
no chasm no a no oapwm no e co oapmm no a
m6 me 0.... me me 3. m6 me 3.

 

.mmSch mg popdoadSa as» am mmapflbfipoc mmeHMoposamnm dam mwmnam

Ioasmslm Hoe consume one: mmagamm paw .wmo pmwSmthsoo who: mcpmpamaomhm map .woa so pmfiooo

AHMOHSU ohms moQSp can .mofiap pmpmoapsa mg» as

.mme Hopes ooom m SH mops» mwswflhpaoo

onwamaoaahaoa ad pmpmom mos was 0.5 mm on poemsnpm was Ads Hem we w.ov osamao one. .Am.m
u apfieapod camaomamv mmpm ommfidmzv psoomm exp smdoaap cmamassa was wmcmfixoposhmosscz

0000 pm omcaHMopozhhossma ho Soapmbapomsa awakens

>H mqmde

 

 

linked assay,

could not be (1
D- and ngluco
tose, D-altros
D-lyxose, D- a
D- and L-ribul
conate, D-gluc'
D-mannitol, D-
“31118 a larger
was determined
at a level of t
the glucose-6-}
abm’e comPound:
066 m D-fruct
lation was com

W
The Nile
tose in the pre

Eben in Table

Effect

\OI‘LH

KinaSe a
Fig. 4.

 

 

28
linked assay, phOSphorylation of the following compounds
could not be detected ((3% of the rate on D-fructose):
D- and L-glucose, Z—deoxyaD—mannose, L—mannose, L-fruc—
tose, D-altrose, D-allose, D— and L-galactose, L-sorbose,
D-lyxose, D- and L—xylose, D- and L—arabinose, D—ribose,
D~ and L-ribulose, D— and L—xylulose, L-rhamnose, D-glu-
conate, D-glucuronate, D-galacturonate, D-glucitol,
D-mannitol, D- and L-arabitol. ribitol, and xylitol. By
using a larger amount of the kinase in this assay, it
was determined that D-glucose was not phOSphorylated even
at a level of 0.04% of the rate with D—fructose. With
the glucose-6-P dehydrogenase-linked assay none of the
above compounds (70 mM) inhibited the phOSphorylation of
0.56 mM D-fructose. indicating that possible phOSphorye
lation was considerably less than the 3% maximum estab=

lished with the other assay.
Phosphoryl Donor Specificity

The relative rates of phOSphorylation of D—frucu
toss in the presence of various nucleotides (3.3 mM) are

given in Table V.

Effect of PH

Kinase activity as a function of pH is shown in
Fig. 4. In different eXperiments, D-fructokinase activ»
ity at pH 6.9 varied from 90 to 100% of D-mannokinase

activity for the same enzyme preparation. The activity

 

 

The nucleo
Mind. The glue
D-fructokinase ac

“4—1:
Nucleotide
\—
ATP

ITP
T'EP
GTP
U’fl’
CT?
IDP
ADP

\

 

29

TABLE V

Phosphoryl donor specificity
of mannofructokinase

The nucleotides were tested at a concentration of
3.3 mM. The glucose-6—P dehydrogenase-linked assay for
Dsfructokinase activity was used.

 

 

Nucleotide Percent relative activity
ATP 100
ITP 75
TTP 62
GTP 9
UTP 9
CTP <4
IDP <4
ADP <4

 

 

 

 

 

 

 

 

Figure 4:

30

pH optima of mannofructokinase. Buf-

fers (0.053 M) used were: cacodylate,
‘ 6.

PH 6.0 to 6.5; glycylgly01ne, pH 9

to 8.0; and glycine, pH 8.5 to 8.9.

0 en—
The pyruvate kinase~lactate dehydr g

th
ass-linked assay was used for bo
shown.
heXOses throughout the pH range
pH 6.9

e—é-P

Activity on D—fructose through

cos
to 8.9 was verified by the slu re-
H measu
dehydrogenase—linked assay. P

reaction
ments were made on duplicate th
wi
mixtures. The pH did not vary

riod.

time during the assay pe

ACTIVITY

RELATIVE

IOO

80

60

40

20

Figure

pi-

 

 

 

 

 

 

31

Figure 4

 

pH-ACTIVITY PROFILE

/7\JX~(‘
80 f/ T\

40 a
D-MANNOSE\ '
2. f Y

 

 

 

 

 

 

 

I\o/

 

RELATIVE ACTIVITY

 

 

 

 

 

 

 

pH

 

 

 

on D-mannose

on D-fructose
around pH 8 t
mannose was a
and about 32%
hexose and ATP
obtained for D4
with the pyruv:
and the glucos
late kinase am
not be detecte<
either the kins
oxidation of NI
With D-fructose
6-P as substrat
genase-linked
pH values. Li
of NADP could
glucose-64J de
at any pH. Th1
profiles were
that the differ
the result of 0
Contribute to o

tides.

The D-m
atDH 6.9. 8.5.

 

32
on D-mannose dropped considerably around pH 8, while that
on D-fructose increased slightly and reached a peak
around pH 8 to 8.5. The rate of phOSphorylation of D-
mannose was about 54% of that of D-fructose at pH 8.5
and about 32% at pH 8.9. The enzyme was saturated with
hexose and.ATP at all pH values. Identical curves were
obtained for D-fructokinase activity from pH 6.9 to 8.9
with the pyruvate kinase-lactate dehydrogenase-linked
and the g1ucose-6-P dehydrogenase-linked assays. Adeny-
late kinase and 6-ph08phofructokinase activities could
not be detected at pH values of 6.9. 7.5. and 8.5 in
either the kinase fraction or the coupling enzymes. No
oxidation of NADH or reduction of NAD could be detected
with D-fructose, D-mannose, Damannose-6-P, or D-fructose-
6-P as substrates in the pyruvate kinase-lactate dehydro-
genase-linked assay mixture minus ATP at the same three
pH values. Likewise, no oxidation of NADPH or reduction
of NADP could be detected with these substrates in the
glucose-6qP dehydrogenase-linked assay mixture minus.ATP
at any pH.fi This indicates that the observed pH-activity
profiles were an eXpression of the kinase activities and
that the differences on D-mannose and D-fructose were not
the result of contamination with other enzymes that might
contribute to oxidation or reduction of pyridine nucleo-
tides.

The D-mannokinase to D-fructokinase activity ratios

at pH 6.9, 8.5. and 8.9 remained constant at various stages

 

 

of pm‘if icati<

eons on disc e

Determination
and D-Fructo se

 

Because
pal velocity u
interest to de
affect the Km
D-mnnose (0.4
that for D-fru
pH 8.9 (Fig. 5
to 4 mid did no‘
at pH 6.9 and a
Burk plot all I

strate axis (F1

    
   
    
    

was 0.4 mM at
D~fructose as
at pH 6.9 and 1
of D-mannose
had no effect 0
8.9 (Fig. 8).

33

of purification, including the fraction that was homogen~
eous on disc electrophoresis.
Determination of Km for D—Mannose
and D-Fructose

Because of the different effects of pH on the maxi-
mal velocity with the two hex0se substrates, it became of
interest to determine whether pH would significantly
affect the Km for D—mannose or D-fructose. The Km for
D-mannose (0.4 mM) was the same at pH 6.9 and 8.9, while
that for D-fructose was 0.4 mM at pH 6.9 and 0.7 mM at
pH 8.9 (Fig. 5). Concentrations of ATP ranging from 0.2
to 4 mM did not affect the apparent Km for either hexose
at pH 6.9 and 8.9. The curves obtained in a Lineweaver=
Burk plot all converged at a common point on the 1/sub-

strate axis (Fig. 6).
Determination of Km for ATP

With D-mannose as the substrate, the Km for ATP
was 0.4 mM at pH 6.9 and 2 mM at pH 8.9 (Fig. 7). With
D-fructose as the substrate, the Km for ATP was 0.1 mM
at pH 6.9 and 1 mM at pH 8.9 (Fig. 7). Concentrations
of D—mannose and D-fructose ranging from 0.2 to 3.3 mM
had no effect on the apparent KIn for ATP at pH 6.9 and
8.9 (F18. 8).

 

 

 

   

 

Figure 5:

34

Lineweaver-Burk plots for determining
the Km values for D-mannose and D-fruc-
toes. The pyruvate kinase-lactate
dehydrogenase-linked assay was used
except that the pH and hexose concen-

trations were varied as indicated.

 

 

35

Figure 5

 

22:5 mmome

_I N:

 

 

 

 

 

N _ O N _ _I N:
A _ _ _ _ _ _\ _ O
mmozzqzs I .x. I _
\
\ . t\ N
\“HD I. O I
h”. ./ .\ _/
o\o\o meRoammIO I mmonbammé I m
nfl‘. 0
I1 .\. I. q
I .\ mmozzgé I m
\
me In. x. mm IQ, .
m x d.

 

 

 

 

 

 

 

Figure 6:

36

Lineweaver—Burk plots showing the rela-
tionship of reaction velocity to D-man-
nose and D—fructose concentrations in

the presence of varying concentrations

of ATP at pH 6.9 and 8.9. The pyruvate
kinase~lactate dehydrogenase-linked

assay was used except that the pH and

ATP and hexose concentrations were varied
as indicated. The numbers along the
curves represent the ATP concentration
(in mM). The Mg++ to ATP ratio was main-
tained at 2:1. A,plot of 1/v vs l/mM
D—fructose at pH 8.9; B, plot of 1/v vs
l/mM Dufructose at pH 6.9; c, plot of UV
vs 1/mM D—mannose at pH 8.9; D, plot of

1/v vs 1/mM D—mannose at pH 6.9.

0)

Figure 6

. FRUCF
DH 89

MANNOE
DH 89

 

 

 

3577

Figure 6

 

 

 

 

 

 

 

 

 

ariefi

pain-

f I/V

7
7. 3 3 7
n/m .«c .,. .«c ..u
”/0 0/0 6/0 II. 2/0/ 6 ”.3
. / I O . 0 ol
0 o o [1 IO 0 o 00 I2
/ / I / I a
Our .m- .fluna .. .w l. .a
a. .o a. a. ”v ..
I I I l a II.
.//. ./.o .I v}
.o a. .x. a. a. a...
an .I .II .IIIIIIII.III.I I .I IIIIII nu
.\O Fr.
m9 - we I
C6 N6 1 _
“WWHHH mm“

Hung pp. nr nun” um NW. nu.
nu n.1r. MW ,Ihmw ,1.. MW “w “WI/l, 1// ac
Illllllnnlllllo. .III.. [I]. III/[I‘r .. o

/OHO /o//o I o, ’0. .0, II
/ / . C C
o o. o. o I]. z. “-
I I I l | I '0
e. a
m9 l 09 ll
nc.uc mm.xc _
mWWHHH mmm
A FD I CMW In...
vI ,hv .no A». ..o A,. II AHV a». .«O “I. .I. Any
II V

 

HEXOSE (mM)

 

 

 

 

 

Figure 7:

38

Lineweaver—Burk plots for determining
the Km values for ATP. The pyruvate
kinase-lactate dehydrogenase-linked
assay was used except that the pH and
ATP concentration were varied as indi-

cated. The Mg++:ATP ratio was main-
tained at 2:1.

 

 

 

39

Figure 7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

22.5 n_._.<

o v e- m- _ N _ O

_ l

\.\L\. _

meRoONmWHVKu \o\ommoeo:E_.o ’

\.
\o\0\o _ o\ . \o A
o @252-.. mmozz<fio o
\ 0

mm IO \.\ mm In
_m _ _ q

 

 

 

 

 

 

 

Figure 8:

40

Lineweaver-Burk plots showing the rela-
tionship of reaction velocity to ATP con-
centration in the presence of varying
concentrations of D-mannose and D-fruc-
tose at pH 6.9 and 8.9. The routine
pyruvate kinase-lactate dehydrogenase-
linked assay was used except that the pH
and ATP and hexose concentrations were
varied as indicated. The numbers along
the curves represent the concentration
(in mM) of the hexose substrate. The
Mg*+IATP ratio was maintained at 2:1.

A, plot of 1/v vs l/mM ATP at pH 8.9
with D-mannose as the substrate; B, plot
of i/v vs i/mM ATP at pH 6.9 with D-man-
nose as the substrate; C, plot of i/v vs
i/mM ATP at pH 8.9 with D-fructose as the
substrate: D, plot of i/v vs i/mM ATP at

pH 6.9 with D-fructose as the substrate.

 

 

 

 

 

 

 

 

 

0
mo.
sud nmo o 1
$6 In ad In
map—.031“. Q mmOhoamu 0
1 and I
4 _
a q _ _ _ A _ J“W\\V
\OVH 0\" 1
. . \\
M“.\u\o \u \ ..
\\\\O\\ J\\ O
. \
va \ So “a o i
no. Qm In . mm In
.0 mmozzsz m 80 mmozzqs < i

 

Figure 8

 

 

 

 

 

 

 

—

Inhibition :
Activity '0: J

The re
fructose were
tive, at pH 6
(Fig. 9) show
tion by D-man
shown in Pig.
to be 0.4 mM,
(Fig. 5).

Product Inhib

 

Inhibi
was tested at
with ATP (Fig
K1 for ADP wa;
at concentrat‘

phorylation 01

Estimation of

 

Data ft
in a sucrose c
ing 3.5 S as ,1
dase standard
mannofructokir

distancez = S;

 

42

Inhibitigg or D.z;gg§ggina§g
ctivitz y D-Mannose

The rates of phoSphorylation of D-mannose and.D-
fructose were found to be competitive, rather than addi-
tive, at pH 6.9, 8.5, and 8.9. A Lineweaver-Burk plot
(Fig. 9) shows the inhibition of D-fructose phoSphoryla-
tion by D-mannose at pH 6.9. From the kinetic plot 3
shown in Fig. 10, the K1 for D-mannose was determined

to be O.h mM, which is the same as its Km as a substrate
(Fig. 5).

 

Product Inhibition

Inhibition of D-fructose phoSphorylation by ADP
was tested at pH 6.9 and was found to be competitive
with ATP (Fig. 11). From a kinetic plot (Fig. 12), the
K1 for ADP was estimated to be 0.3 mM. D-Fructose-6qP,
at concentrations up to 15 mM, did not inhibit the phos-
phorylation of D-fructose at either pH 6.9 or pH 8.9.

Estimation of Molecular Weight

Data for the sedimentation of mannofructokinase
in a sucrose density gradient are shown in Fig. 3. Tak-
ing 3.5 S as the sedimentation coefficient of the peroxi-
dase standard (23), the sedimentation coefficient of
mannofructokinase was calculated by the equation 31 x
distancez = 32 X distancel (2#) to be 4.1 8. Assuming

:4 ,1 ”A

 

 

 

 

 

 

Amt.
I .

 

Figure 9:

43

Lineweaver-Burk plot showing the relation-

ship of reaction velocity to D-fructose
concentration in the presence and absence
of D-mannose. The glucose-é-P dehydro-
genase-linked assay for D-fructokinase
activity was used except that D-fructose
and D-mannose were varied as indicated.

The pH was 6.9.

 

 

 

41»

Figure 9

 

SE owopofihmlm
.H

AV .H.. mm..

 

 

mmOGQMEIQ 25 O

O
onoaaoano 2a m.a

HF

 

 

 

ition-
as
sence

['0-

tose

;ed.

 

 

 

 

 

 

 

fink

AD‘KM.
uA

 

Figure 10:

45

Kinetic plot for determining the K1
for D-mannose. The data are taken

from the eXperiment described in

Figure 9.

20

IS

-.<|—

IO

46

Figure 10

 

K. FOR o-MANNOSE/

 

 

 

 

=O.4 mM /0

 

l
|.3 mM

 

 

./

 

0.33 mM D-FRUCTOSE

D-FRUCTOSE

__1\ ./_.

 

\:

 

'\

 

 

A 2.7 mM o-FRUCTOSE

 

 

-l o I
D-MANNOSE (mM)

2

 

 

 

 

Figure 11:

47

Lineweaver—Burk plots showing the rela-
tionship of reaction velocity to ATP

concentration in the presence of vari—

ous concentrations of ADP. The glucose—'

64F dehydrogenase-linked assay was used
except that ATP and ADP were varied as
indicated. The Mg++ concentration was
maintained at twice the total concentra—

tion of ATP plus ADP. The pH was 6.9o

Figure

 

 

28

rela-

'ari-

.UCOSE'

; used
ad as

l was

:entra-

Figure 11

 

  

() {mid 1313]”

 

 

 

 

ZEES

221+ -—-

22C)

-23

-4

-6

 

Inli

IXGTI’

 

 

 

 

Figure 12: Kinetic plot for determining the K1
for ADP. The data are taken from the

eXperiment described in Figure 11.

5O

 

 

Figure 12

 

 

mew SS mm.o

\\ o

 

 

 

 

in

NH

ma

ON

3N

mm

‘4';

 

that the man

its molecule
of log S ver
Tanford (25)
approximate]
sedimentatio

The p
reactions we
Sul (0.06 11
obtained fro:
strate (D-fr
uncle of MgC
glycine buff
0.15 ml. Af‘
addition of I
caused no in
tose as the
of glucose-6
increase at
umole of D-g
With D-manno
0.0048 unit
the cuvette

absorbance

lent to the

 

 

51
that the mannofructokinase molecule is roughly Spherical,
its molecular weight was estimated with the aid of a plot
of log S versus log molecular weight, using the data of
Tanford (25), This method gives a molecular weight of
approximately 47,000 for the eXperimentally determined

sedimentation coefficient of 4.1 8.
Product Identification

The products of the mannofructokinase—catalyzed
reactions were prepared by incubating in a microcuvette:
5 ul (0.06 unit) of the homogeneous enzyme preparation
obtained from disc electrophoresis, 0.02 umole of sub-
strate (D-fructose or D-mannose), 0.5 umole of ATP, 1.0
umole of MgClZ, 0.1 umole of NADP, 8.0 umoles of glycyl-
glycine buffer (pH 6.9). and water to a final volume of
0.15 ml. After incubation at 25°C for one hour, the
addition of excess glucose-é—P dehydrogenase (0.078 unit)
caused no increase in absorbance at 340 nm. With D-frucu
tose as the substrate, the further addition of 0.31 unit
of glucose—é—P isomerase resulted in an absorbance
increase at 340 nm equivalent to the oxidation of 0.022
umole of D—glucose-é-P; no change was noted in the cuvette
With D—mannose as the substrate. The further addition of
0.0048 unit of mannose-é-P isomerase caused no change in
the cuvette with D-fructose but resulted in a 340 nm
absorbance increase in the cuvette with D-mannose equiva-

lent to the oxidation of 0.021 uncle of D-glucose—é-P.

 

 

Assay

dehydrogenas
in the kinast
negative. Tl
phOSphorylatf

tose-6-P and

 

 

 

52
Assays for possible contaminating 6—phoSphogluconate
dehydrogenase, phoSphofructomutase, or phOSphomannomutase
in the kinase preparation and in the coupling enZymes were
negative. These results indicate that the products of the
phoSphorylation of D—fructose and D-mannose were D—fruc—

tose-64F and D-mannose-é-P, resPeotively.

 

 

 

Previ
nose or D-fr
D-glucose an
efficiencies
hexose (9-12
ent in that :
which is an a
(D-fructose) ‘
other sugars
basis for thi
tural formula
tion of molec
31.6% in the
sidered to be
tion that the
in their opti
a-D-mannopyra'
fen-ed oonfon
tofuranose an
the positions
models are su]

model are skew

positional co:

 

 

DISCUSSION

Previously reported hexokinases active on D-man-
nose or D—fructose at carbon atom 6 also phOSphorylate
D—glucose and often several other sugars with varying
efficiencies (26—35), or are Specific for a single
hexose (9-12). The hexokinase described here is differ-
ent in that it is equally active on two hexoses. one of
which is an aldose (D~mannose) and the other a ketose
(D-fructose), but has no detectable activity on many
other sugars (<0.04% in the case of D—gluCOse). The
basis for this Specificity is not apparent from struc-
tural formulas but can be rationalized from an inspec-
tion of molecular models. D-Fructose in solution occurs
31.6% in the furanose form (36) and is generally con-
sidered to be the B anomer (37), presumably on the assumpa
tion that the a and B anomers would differ significantly
in their optical rotations. D-Mannose in solution is 69% I
G-D-mannopyranose (38). Molecular models of the pre—
ferred conformations of d-D—mannopyranose and B-D-fruc-
tofuranose are depicted in Fig. 13. It can be seen that
the positions of the oxygen and hydrogen atoms on the two
models are superimposable, although the positions on one
model are skewed somewhat relative to the other. This

positional correSpondence of equivalent atoms holds even

53

 

 

 

   

 

 

 

 

 

Figure 13:

 

54

Structural models showing the topological

similarity between d—D-mannopyranose (left

side of each photograph) and B-D—fructo—

furanose. Upper, bottom view of the
models; lower, top View of the models.

Carbon atom 6 is located at the bottom

of each photograph-

 

 

Figure 13

 

 

though one
and the oth
D-mannose a
ity which i
sugars test
enzyme has
strate site
not by both
other sugar:
Moon
SEW
bacterium, ¢
Specific 13-1
heXOkinase e
KinaSes Obta
6'Pho'sphoryl
D‘Slucose a1
“her (9-12)
derlGe that a
responsible
D‘fructose, .
for the mo ‘
PUrified to ;
electrODhore;
of the W0 a«
separation We

acetate st“!

56
though one of the models consists of a five-membered ring
and the other a six-membered ring. Viewed in this way,
D-mannose and D-fructose bear a close structural similar-
ity which is not mimicked by D-glucose or any of the other
sugars tested as possible substrates. Thus, although the
enzyme has strict requirements for binding at the sub-
strate site, it can be seen how these conditions may be
met by both Dumannose and D-fructose to the exclusion of
other sugars.

Moore and O'Kane (11) presented evidence that
Streptococcus faecalis, a homofermentative lactic acid
bacterium, contains a specific D-mannokinase and a
Specific D-fructokinase in addition to a nonSpecific
hexokinase active on Duglucose, D-mannose, and D-fructose.
Kinases obtained from other sources which catalyze the
6-ph08phorylation of D-mannose or D-fructose but not
D-glucose also seem to be Specific for one hexose or the
other (9-12). Thus, it is important to review the evi-
dence that a single enzyme from‘g. mesenteroides is
responsible for the phosphorylation of both D-mannose and
D-fructose, particularly since the pH-activity profiles
for the two hexoses are different. The enzyme has been
purified to apparent homogeneity (determined by disc gel
electrophoresis) with no significant change in the ratio
of the two activities throughout the purification. No
separation was achieved by electrophoresis on cellulose

acetate strips or by sucrose density gradient centrifuga-

 

 

tion.
value
ally,
tive :

the s

and D-

'pect 1

be n01
bit di
examp]
Eenase

(28).

fructo
kinase
data p
it? of
Streng-
in 0.1

f°r mo:

t°k1na:
Of One
Km of t

°f Subs

 

 

57
tion. Thermal inactivation rates assayed at three pH
values were identical for the two activities. And fin-
ally, the phOSphorylation of the two hexoses was competi-
tive rather than additive, with the Ki for D-mannose being

the same as its Km as a substrate.

 

The differential pH-activity profiles for D-mannose
and D—fructose are of interest and at first led me to sus~
'pect that two enzymes were involved. However, it should
be noted that several other enzymes are known which exhi-
bit different pH optima for different substrates, for
example, fructose diphOSphatase (39), glutamate dehydro-
genase (40), and hexokinase from ASpergillus parasiticus
(28).

The common identity of the D-mannokinase and D-
fructokinase activities and the instability of D—gluco-
kinase of L. mesenteroides were recently corroborated by
data presented by DeMoss (14). He attributed the labilu
ity of D—glucokinase observed by us to the low ionic
strength of our cell extracts. His enzyme preparation
in 0.1 M potassium phosphate buffer (pH 7.5) was stable
for months at -20°C and for days at 0°C (14).

Although the reaction mechanism of this mannofruc-
tokinase has not been studied in detail, the inability
of one substrate (hexose or ATP) to affect the apparent
Km of the other is consistent with a sequential mechanism

of substrate binding (41), as for yeast hexokinase (42-44),

 

 

. ”Hui:

(T. 4:wa
r-)

 

 

 

58

E. 22;; galactokinase (45), and rat muscle hexokinase
type II (46), rather than with the "ping—pong" type (41),
as is characteristic of nucleoside diphoSphate kinase
(47, 48) and rat muscle hexokinase type I (49). Earlier
studies on particulate (50) and solubilized (51) brain
hexokinase suggested that these enzymes exhibit a "ping—
pong" mechanism of action. Recent data, however, indi-

cate that the above mechanism is incorrect, and that the

mechanism appears to be sequential (52. 53).

 

 

SUMMARY

An adenosine 5'-triphOSphate:hexose 6-phOSpho-
transferase Specific for D-mannose and D-fructose (manno-
fructokinase) was purified to apparent homogeneity from
extracts of‘pguconostoc mesenteroides. D-mannose and
D-fructose were phoSphorylated by the enzyme at equal
rates, whereas D-glucose, 2-deoxy-D-mannose, and 28
other sugars and sugar derivatives were not phoSphory-
hated and did not inhibit the enzyme. The pH-activity
curves were different for D-mannose and D-fructose, with
the Demannokinase activity to D-fructokinase activity
ratios being about 1.0 at pH 6.9, 0.5 at pH 8.5, and 0.3
at pH 8.9. The enzyme was further characterized with
regard to phOSphoryl donor Specificity, kinetic constants,
inhibition constants, and molecular weight. The products
of the phoSphorylation of Demannose and D-fructose were
identified as D-mannose-6-P and D-fructose-é-P, reSpec-
tively.

To explain the unique Specificity of this kinase,
it was postulated that d-D-mannopyranose and B-D-fructo-
furanose are the molecular Species that serve as sub-
strates. It was shown with molecular models that equiva-
lent functional groups of the preferred conformations of

the two Species occupy nearly identical Spatial positions,

59

 

 

 

60
even though one of the molecules consists of a five—
membered ring and the other a six-membered ring. Viewed
in this way, D-mannose and D—fructose bear a close struc—
tural similarity which is not mimicked by D-glucose or
any of the other sugars tested as possible substrates.
Thus, although the enzyme has strict requirements for
binding at the substrate site, it can be seen how these
conditions may be met by both D-mannose and D-fructose

to the exclusion of other sugars.

 

 

 

.ricb

 

PART II

l-PHOSPHOFRUCTOKINASE AND 6-PHOSPHOFRUCTOKINASE
FROM AEBOBACTER AEROGENES

INTRODUCTION

The discovery in 1966 of an inducible kinase

_:—*.—- —«-A-------|-i

Specific for D-fructose l-phosphate in Aerobacter

i am?- Hi.

aerogenes PRL—RB (54) suggested a previously unrecog-
nized pathway of D-fructose metabolism. More recently,
a four-component PEP:fructose l-phoSphotransferase sys-
tem from this organism was characterized and genetic
evidence was‘presented for the requirement of the
enZyme system for normal growth on D-fructose (55).
This work thus established the pathway for D-fructose
metabolism to be the following:

PEP system 1—PFK
D-Fructose ’3»D-fructose-i-P ; FDP

 

 

In addition to the PEP system and 1-PFK, A,
aerogenes PRL—R3 has also an inducible D-fructokinase
(ATPxD-fructose 6-phOSphotransferase) and a constitutive
6-PFK. An alternate route,

D-fructokinase 6-PFK
D-fructose I; D-fructose-6-P ————_—9 FDP,

 

may therefore be operative in this organism. Section A
of this part of the thesis assesses the relative impor-

61

 

 

:l. .59“
I
D
,"
‘ |

 

 

62
tance of these two pathways during growth on D-fructose.
Analysis of mutants lacking 6-PFK and FDPase corroborate
the view that D—fructose is metabolized via the D-fruc-
tose—i-P pathway and establishes that 6-PFK is functional
in the metabolism of D-glucose but not D-fructose. This
work has been published recently (56).

The presence in A. aerogenes of two phOSphofructo—
kinases with different roles makes it of interest to
study the two enzymes and compare their control mechan~
isms. FDP, the product of the 6-PFK reaction, can be

converted back to D-fructose~6—P by FDPase:

ATP ADP
6-PFK
D-fructose-é-P FDP
FDPase

The D-fructose—6-P—FDP cycle functions as a net ATPase
if not controlled; hence it has been termed a "futile"
Cycle (57). l-PFK, on the other hand, is not known to
participate in such a cycle. It is therefore to be
eXpected that 6—PFK would be subject to more complex
control mechanisms than would l-PFK. 6-PFK from a
variety of organisms is being intensively studied with
respect to its regulation (see Review of Literature in
Part II, Section B). l-PFK, on the other hand, which

is now known also to occur in Bacteroides symbiosus (58)

and Escherichia coli (59), has not previously been sub-

 

 

 

 

63
jected to kinetic analysis. The last portion of this
thesis (Part II, Section B) presents evidence that the
6-PFK from A. aerogenes, like those from most other
organisms, diSplays sigmoidal kinetics and is modified
by several effectors, whereas the l—PFK exhibits regu-
lar MichaeliS-Menten kinetics and more closely resembles

other non-allosteric kinases.

 

 

SECTION A

DETERMINATION OF THE RELATIVE SIGNIFICANCE OF
D-FHUCTOSE-l-P AND D-FRUCTOSE-6-P PATHWAYS IN
5. AancENEs BY ANALYSIS OF MUTANTS
LIEKINE'EZPHoSPHOFRUCIOKINASE
AND D-FBUCTOSE 1,6-DIPHOSPHATASE

MATERIALS AND METHODS
Bacteria

The parental strains used in this investigation
were Aerobacter aeroggnes PRL-R3 and a uracil auxotroph,
PHL-HB(U'), derived from it. The uracil auxotroph was
given to us by Dr. Robert P. Mortlock of the University
of Massachussetts. Mutant 012, derived from strain
PRIPBB, was isolated by T. E. Hanson (56); mutant A9-1,
derived from strain PHL-H3(U'), was isolated by Dr. H.
L. Anderson (56).

Culture Media

The basal mineral medium used for strain PBL-B3
and mutant 012 consisted of 0.71% NaZHPou, 0.15% KHZPOu,
0.3% (NH4)2304, 0.009% MgSOn, and 0.0005% FeSOu°7H20.
This medium was supplemented with 0.005% uracil for the
growth of strain PBL-R3(U') and mutant A9-1. Sugars
were autoclaved separately and added to the basal mineral
medium at a concentration of 0.5%.

64

 

65
Growth of Cells and Preparation

of Extracts

The growth curves were done in 18 x 50 mm culture
tubes containing 7.0 ml of medium. The inoculum was 0.1
ml of an overnight culture on D-glucose (except for
mutant A9-1, which was on D-fructose). The tubes were rm-
slanted at an angle of 55° and were agitated on a water

bath reciprocal shaker at 148 cycles per min at 30°C.

For enzyme studies, the cells were grown in 500

If. Him—‘1‘“- — -4. 1.12..

ml of medium in Fernbach flasks on a rotary shaker at
32°C. The cells were harvested by centrifugation during
the late log phase of growth, suSpended in distilled
water, and broken by sonication for 10 minutes as des-

cribed in Part I.
Chemicals

FDP and crystalline a-glycerophOSphate dehydro-
genase-triose phoSphate isomerase were from Sigma. D-
fructose-6-P was from Boehringer. D-fructose-l-P, yeast
glucose-6-P dehydrogenase (A grade), rabbit muscle FDP
aldolase (A grade), and crystalline rabbit muscle glu-
cose-6-P isomerase (A.grade) were from Calbiochem. All

other chemicals were obtained as described in Part I.

Enzype.Assays

All assays involved the oxidation or reduction of

pyridine nucleotide coenzymes and were monitored at 340 nm

 

 

66
with a Gilford automatic absorbance-recording spectro—
photometer thermostated at 25°C. The reactions were
carried out in 0.15—ml volumes in microcuvettes with a
1—cm light path. In all cases, the amount of extract
assayed was limiting, so that the rates were propor-
tional to the enzyme concentration. Specific activity
was defined as the number of umoles of substrate uti-
lized per minute per milligram of protein.

The assays for i-PFK and 6-PFK contained 1.0
uncle of ATP; 2.0 mmoles of MgClZ; 0.05 uncle of NADH;
1.0 umole of D-fructose—l-P or D—fructose—6—P; excess
FDP aldolase, triose phOSphate isomerase, and a-glycero-
phoSphate dehydrogenase; and 10.0 umoles of buffer
[glycylglycine (pH 7.5) for l-PFK, and glycine (pH 8.2)
for 6—PFK]. The control assays contained all components
of the reaction mixture except ATP.

The assay for FDPase contained 1.0 umole of FDP,
1.0 umole of MgClz, 0.2 umole of EDTA, 0.1 uncle of NADP,
exoess glucose-é-P isomerase and glucose-6—P dehydrogen—
ase, and 10.0 mmoles of glycylglycine buffer (pH 7.5).

The assay for D-fructokinase activity contained
1.0 umole of D-fructose, 0.5 uncle of ATP, 1.0 umole of
M8012, 0.1 umole of NADP, excess phOSphoglucose isomerase
and glucose~6-P dehydrogenase, and 10.0 mmoles of glyoyl-
glycine buffer (pH 7.5).

The assay for D-glucokinase, as described by

Kamel, Allison, and Anderson (17), contained 1.0 umole

 

 

 

67
of D—glucose, 0.5 umole of ATP, 1.0 mmole of MgClz, 0.1.
umole of NADP, excess glucose-6-P dehydrogenase, and

10.0 umoles of glycylglycine buffer (pH 7.5).

Protein Determination

Protein was estimated as described in Part I.

 

 

RESULTS
Growth Pattern

Growth characteristics of the parental strain
(PRL-Rj) and the two mutants (A9—l and 012) on D—glucose,
D-fructose, and glycerol are shown in Fig. 14. Strain
PBL—H3 grew well on all three substrates. Mutant A9-1
mimicked the parent on D-fructose and glycerol, but
grew only slowly on D-glucose. Mutant 012 grew well on
D-glucose but failed to grow on D-fructose or glycerol;
after 24 hr, slight growth occurred occasionally on D—

fructose but not on glycerol.
Enzyme Activities in Cell Extracts

The data in Table VI Show that all strains con-
tained similar levels of D—glucokinase, whereas mutant
012 was missing FDPase and mutant A9-1 was missing 6-PFK.
D—Fructokinase activity was low in all extracts, but was

consistently higher in cells grown on D—fructose than on

 

D-glucose. This apparent Dwfructokinase activity has
not been purified, so it has not been established that
the observed activity in crude cell extracts is the
result of a single enzyme possessing ATPzD-fructose 6-
phOSphotransferase activity. The presence, however, of

a phOSphofructomutase in the extract and the coupling

68

 

 

”1:15

 

 

Figure 14:

69

Growth characteristics of strain PRL—H3
and mutants 012 and A9-1 on D-glucose,
D-fructose, and glycerol. The growth
pattern of PHL-H3(U') (not Shown) was
the same as that for PBL-RB. An Optical
density of 0.35 was equivalent to a

viable count of 8.2 x 108 cells per m1.

70

Figure 14

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72
enZymes under the assay conditions was ruled out since
the replacement of D—fructose plus ATP with 1.0 uncle
of D-fructose-l-P in the assay gave no detectable reac-
tion; thus the product of the D~fructokinase reaction

was shown to be D-fructose-6-P.

 

1-PFK was found only in extracts of cells grown
on D-fructose (Table VI). The inability of mutant 012
to grow on D-fructose precluded a measurement of 1-PFK

in this strain under conditions which induced the enzyme

p..-

n the other strains. However, partial induction was
achieved by incubating D—glucose-grown cells in 0.25%
D-glucose plus 0.25% D-fructose in mineral medium for a

period of time sufficient to allow complete utilization

 

 

of the D-glucose. Under these conditions, the specific
activities in extracts were 0.12 in strain PRL-BB and
0.013 in mutant 012. When the cells were harvested and
extracts prepared before D-glucose utilization was com~
plete, 1-PFK activity remained undetectable, indicating
repression in the presence of nglucose. The partial
induction observed in mutant 012 probably occurred in
the short period of growth just before or immediately
after the D—glucose was exhausted and D-glucose repress
sion was relieved. A further attempt was made to
induce 1-PFK in mutant 012 under conditions in which
D-glucose repression would be absent by SXposing D-
glucose—grown cells to 0.25% D—fructose in nutrient

broth (0.5% Difco peptone plus 0.3% Difco beef extract,

 

 

73

pH 7.0). l—PFK activity remained undetectable in mutant
012, although a normal level of activity was induced in
strain PRL—R3. This lack of induction in mutant 012
may be attributed to catabolite repression, which is
known to be enhanced during catabolism under nongrowing
conditions (60, 61); mutant 012 does not grow on nutri-
ent broth, which is consistent with its lack of FDPaSe.

FDPase activity as a function of assay pH is
shown in Figure 15. The extract from mutant 012 exhibited
some activity at low pH values, but no activity at pH
7.5, which was the pH optimum for FDPase activity in
extracts of strain PRL—H3 and mutant A9-1. The activity
at low pH values is believed to be due to a nonSpecificr

acid hexose phoSphatase (62).

 

 

 

 

 

"it-.5

 

Figure 15:

7c

pH-Activity profile of FDPase. The
standard assay was used except that
the buffer composition and pH were
varied. Buffers (10 mmoles) used
were: cacodylate, pH 5.0 to 6.5;
glycylglycine, pH 7.0 to 8.0; and
glycine, pH 8.5. The profile for
A9-1 (not Shown) was the same as

that shown for PHL-HB.

UNITS/GRAM OF PROTEIN

4O

3O

20

IO

75

Figure 15

 

o-FiaucIOSI'a l,6-DI(3HOSPr-)ATASE

 

 

 

 

 

 

 

 

 

 

 

 

 

’I ' ini_r.--l- -.“ ’

DISCUSSION

The possible common pathways for the metabolism
of D—glucose, D—fructose, and glycerol in A. aerogenes
are as summarized in the scheme shown in Fig. 16. The
metabolism of D-glucose through the Embden-Meyerhof path-
way requires 6-PFK. If D—fructose were metabolized via
D-fructose-6-P, then it follows that 6-PFK would also
be required for normal growth on this substrate. On the
other hand, if it were metabolized through D-fructose-
l-P, then the 6—PFK—catalyzed reaction would be bypassed.
In the latter case, normal growth on D—fructose would
require FDPase to make D-fructose-6-P for biosynthetic
reactions. Normal growth on glycerol would likewise
require FDPase.

Mutant A9—1, missing 6—PFK, grows on D-fructose
or glycerol as well as does the parental strain, PBL-RB
(U'). but grows only slowly on D—glucose. This is con-
sistent with the metabolism of D-fructose through D—
fructoseAI-P. If fructose were metabolized through D—
fructose-6-P rather than D—fructose—l—P, this mutant
would still be SXpected to grow Well on glycerol. but
no better on D—fructose than on D-glucose. The residual
growth on D-glucose by this mutant could indicate that

the defective 6-PFK is partially functioning in the

76

 

 

 

 

 

77

Figure 16: Pathways for the metabolism of
D-glucose, D-fructose, and

glycerol in A. aerogenes.

Jill“

78

 

Figure 16

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79

intact cell, but is more likely due to the metabolism
of D-glucose through the hexose monophOSphate shunt.

Mutant 012, missing FDPase, grows well on D-
glucose but not on D-fructose or glycerol. This, too,
is consistent with D-fructose being metabolized in the
wild type primarily through D-fructose-i-P rather than
D-fructose-é-P. If the pathway through D-fructose-6AP

; tum . a ’U1

were of major Significance, a FDPase-negative mutant

would be expected to grow on both D-glucose and D-fruc-

 

tose.

Similar mutant analysis has recently been carried
out on E. gg_]_._1_ to assess the relative importance of the
D-fructose-iAP and the D-fructose-6-P pathways in D-
fructose metabolism of this organism (59, 62, 63).
Although an earlier paper indicated that the metabolism
goes through D-fructose-6-P (62), more recent results
(59) indicate that the D-fructose-i-P pathway occurs
also in.§, gglg. However, some mutants of this organism
deficient in 6-PFK activity failed to grow normally on
D-fructose, suggesting that 6-PFK may have some role in
Dbfructose metabolism (63). The identification of a
PEPzD-fructose 1-phOSphotranSferase system and a iéPFK

in this organism provides support for the D-fructose-i-P

pathway (59).

Ase-b

 

 

 

 

SECTION B

PURIFICATION, PROPERTIES, AND REGULATION OF
6-PHOSPHOFRUCTOKINASE AND l—PHOSPHOFRUCTOKINASE

REVIEW OF LITERATURE

The central role of 6-PFK in the control of gly-
colysis is well recognized (57, 64—67). lg MAKE SXperi-
ments determining metabolite flux during glycolysis haVe
indicated that the main rate-controlling step is the
6-PFK-catalyzed production of FDP from D-fructose-6-P

(68, 69). Studies of the kinetic properties of the

 

enzyme provide important information on the possible
mechanism for its control. Table VII gives a summary
of most of these studies. Although some results varied
with the system under investigation, it seems clear
that the enzyme is subject to a number of complex con-
trol mechanisms. In all cases [except with the 6—PFK

from Dictyostelipm discoidepm (91)], the enzyme is

 

inhibited by high levels of ATP. In some studies, how-
ever, it is not certain whether the inhibition is a
function of the amount of ATP pg; pg or to the lack of
sufficient Mg++ to bind. all the ATP molecules in a
MgATP complex (84, 89, 90), which is the real substrate
Of the reaction (113). With several mammalian 6-PFKS
significant ATP inhibition was detected under conditions
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97
of excess Mg++ (71—75, 80—83, 92-94), indicating that the
MgATP complex can bind to a regulatory site and inhibit
the reaction. Equilibrium binding studies by Kemp and
Krebs (98) on skeletal muscle 6-PFK have indicated that
three moles of ATP are bound per 90,000 g of the enzyme.
which is probably the molecular weight of the protomer
(77). g. 32;; 6-PFK is inhibited by ATP only when the
Mg++ to ATP ratio falls below 2:1 (95, 96). Blangy gt
a; (97) failed to detect ATP inhibition at a constant
Mg++ to ATP ratio of 1021..

Increased levels of D-fructose—o-P generally
relieve ATP inhibition (70-74, 78, 80, 82, 83, 86, 88-90,
92-96). Exceptions are the 6-PFKs from cockroach (85)
and calf lens (8b). The rates of most 6-PFKs studied
(71-74, 78, 80, 82, 83, 86, 88~90, 92—96) show a sigmoidal
dependence on the D-fructose-6-P concentration. E. 32;;
6-PFK also shows a sigmoidal dependence of rate on the
Mg++ concentration; increasing the D-fructose-é-P con-
centration from 1.2 mM to 4.8 mM serves to convert the
sigmoidal curve to a hyperbolic one (95).

6-PFK is generally non—specific with regard to
its phOSphoryl donor (71, 72, 78, 88, 91-95). Further,
several mammalian 6-PFKs are inhibited by other nucleo—
side triphosphates (71, 73, 78) in a manner similar to
ATP inhibition. In yeast (92—94) and E. 32;; (95),

however, other nucleoside triphOSphates either inhibit

 

 

 

98
the enzyme very slightly or not at all even at low Mg++
concentrations.

Mammalian 6-PFKs in general have more positive
effectors than those from plants or bacteria. AMP and
cyclic AMP, which can relieve ATP inhibition of many
:mammalian 6-PFKs (71-75, 78, 80-82, 84) either have no
effect (90-97) or even inhibit activity (88-90) of the
enzyme from several plants and bacteria. ADP relieves
.AT? inhibition to various degrees in mammalian (71, 72,
7h, 78, 80, 81, 84) and bacterial (90, 95, 96) 6-PFKS
but was found to inhibit the enzyme from several plants
(88-90).

Some mammalian 6-PFKs have been reported to be
subject to control by pH. Calf lens (89) and rabbit
muscle (73, 75) 6-PFKs are considerably inhibited at
pH values near 7, but are inhibited very slightly or
not at all at pH 9. In a similar manner, the enzyme
from rat diaphragm (99) is more susceptible to ATP
inhibition at pH 7.1 than at pH 7.6. Sheep heart 6-PFK
exhibits regular Michaelis-Menten kinetics at pH 8.2
with reSpect to ATP, Mg++, or D-fructose-é-P, but shows
sigmoidal kinetics with respect to D-fructose-é-P at
pH 6.9 (72). Increased concentrations of D-fructose-6-P
shift the pH Optimum of frog muscle 6-PFK towards the
acid side, and this effect is further enhanced by the

presence of AMP (100).

 

 

“11

99
The only 6-PFK reported so far in the literature
' to exhibit non-sigmoidal kinetics is that from Dictyostelium

discoideum (91). This enzyme is not inhibited by ATP,
and is not activated by D-fructose-é-P, AMP. or P1.
.ADP, PPi, and FDP inhibit activity.

In addition to g, aerogenes. l-PFK has now been
reported to occur in Bacteroides symbiosus (58) and
g. 931.; (59). However, no kinetic studies of this enzyme

have yet been published.

MATERIALS.AND METHODS
Bacteria

The organism used for most of the studies on 6—
ZPFK is the wild-type.Aerobacter aerogenes PRL-HB.
Idutant DD31, lacking i-PFK activity, was used for the
inhibition study with D-fructose-l-P; this mutant was
derived from the wild-type strain and was isolated by
if. E. Hanson by the same procedure as for mutant 012
(56). Mutant A9-1, lacking 6-PFK activity. was used as
the source of i-PFK; this mutant was derived from the
‘parental strain PHL-R3(U') and was isolated by Dr. R. L.
.Anderson (56).

Gzowth of Cells and
greparation of Extracts

The components of the mineral salts medium are
as described in Part II. Section A. Strains PRL-HB and
IDD31 were grown in 500 ml of glucose-mineral salts
:medium in Fernbach flasks onia rotary shaker at 32°C.
The cells were harvested by centrifugation at 16,300 x
g in a Servall refrigerated centrifuge.

mutant A9-1 was grown in 100 liters of D-fructose-
mineral salts medium supplemented with 0.005% uracil in

a New Brunswick Model 130 Fermacell fermenter at 30°C

100

 

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101

with an aeration rate of 6 cubic feet per minute and an
agitation speed of 300 rpm. Dow Corning antifoam B
(0.02%, sterilized separately) was added. The inoculum
was 2 liters of an overnight culture in D-fructose-
uracil-mineral salts medium. The cells were harvested
with a Sharples AS-12 centrifuge 8 hr after inoculation.
The yield was about 7.5 g (wet weight) of cells per
liter.

Cells were washed once with 0.01 M Tris-HCl-0.03 M
NaCl (pH 7.3) and centrifuged at 16.300 x g. Mutant A9-1
was taken up in water, while strain PHL-R3 and mutant
D031 were taken up in 0.05 M phOSphate buffer (pH 7.5)
containing 0.001 M EDTA (for the reason for the use of
phOSphate buffer and EDTA, see Properties, section on
Stability). The resulting suSpension was subjected to

sonic oscillation for 10 min as described in Part I.
Chemicals

Crystalline a-glycerophOSphate dehydrogenase and
triose phOSphate isomerase were from Calbiochem. Pig
heart malic dehydrogenase and twice crystallized yeast
alcohol dehydrogenase were from Worthington. D-Fructose-
14F, D-glucose-l-P, D-glucose-é-P, horse heart cytochrome
C. and all nucleotides (except ATP) were from Sigma.
L-Fructose-i-P was the preparation described by Mayo and
Anderson (101). hCaloium phOSphate gel was prepared by

D. P. Allison of this laboratory according to the method

 

 

 

 

 

 

102
described by 0. Levin (102). All other chemicals were

obtained as described in Part I or Part II, Section A;
Enzyme Assays

The routine assay for 6-PFK was as described in
Part II, Section A, except that glycylglycine buffer
(pH 8.0) was used. The aldolase-linked assay for 1-PFK
was as described in Part II, Section A. The pyruvate
kinase-lactate dehydrogenase-linked assay for l-PFK was
used for substrate Specificity and FDP inhibition
studies. The reaction mixture (0.15 ml) contained:

1.0 umole of D-fructose-i-P, 0.5 umole of ATP, 1.0 umole
of MgClZ, 0.# umole of PEP,0.05 uncle of NADH, 10.0 umoles
of glycylglycine buffer (pH 7.5), excess lactate dehydro-
genase and pyruvate kinase, and limiting amounts of

l-PFK. The assays were done in microcuvettes with a

1-cm light path. The rate of oxidation'of NADH was
measured at 340 nm in a Gilford multiple sample absorbance-
recording Spectrophotometer thermostated at 25°C.

In all cases, the rates were directly proportional
to the enzyme concentration. The rate of NADH oxidation
with the aldolase-linked assay for 1~PFK was double that
obtained with the pyruvate kinase-lactate dehydrogenase-
linked assay. A.unit of activity was defined as the
number of umoles of substrate phOSphorylated per minute.

Specific activity was defined as the number of units per

milligram of protein.

 

 

 

 

 

 

 

103

The assay for mannitol-l-P dehydrogenase was the
same as the routine assay for 6-PFK, except that ATP was
omitted from the reaction mixture. The assay for FDPase
was as described in Part II, Section A. The assay for
adenylate kinase was the same as the aldolase-linked
assay for 6-PFK or i-PFK, except that ATP was replaced
with 1.0 umole of ADP.

The assays for malic dehydrogenase and alcohol
dehydrogenase were done in microcuvettes with a 1-cm
light path and monitored at 340 nm with a Gilford Spec—
trophotometer. The reaction mixture for alcohol dehydro-
genase contained, in a volume of 0.15 ml: 10 umoles Of
ethanol, 0.1 umole of NAD, 10.0 umoles of glycylglycine
buffer (pH 7.5). and limiting amounts of alcohol dehydro—
genase. The reaction mixture for malic dehydrogenase
contained, in a volume of 0.15 ml: 1.0 umole of oxalo-
acetate (neutralized to pH 7.0), 0.05 umole of NADH,

10.0 mmoles of glycylglycine buffer (pH 7.5), and limit~

ing amounts of malic dehydrogenase.
Protein Determination

Total protein was estimated as described in Part I.

Cytochrome c was determined by measuring absorbance at

550 nm.

 

 

 

 

 

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RESUDTS
Purification of 6-Ph08phofructokinase

.All Operations were performed at O-hOC. A.sum-
mary of the purification procedure is given in Table

VIII.
Protamine Sulfate Precipitation

The protein concentration of the crude extract
was adjusted to in mg per ml. After the addition of
solid ammonium sulfate to a final concentration of 0.2 M,
the nucleic acids were precipitated by the slow addition
of 20% by volume of a 2% aqueous solution of protamine
sulfate (pH 7.0). The precipitate obtained by centrifuga-

tion was discarded.
Ammonium Sulfate Fractionation

Solid ammonium sulfate was added slowly with
stirring to the above fraction. The precipitate that
formed between 0 and 60% saturation was collected by
centrifugation and dissolved in 0.05 M sodium phosphate
buffer-0.001 M EDTA (pH 7.5).

Sephadex 0-200 Chromatography

A 1-ml aliquot of the ammonium sulfate fraction

104

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106
was placed on a 23.5 x 1.2-cm column Of Sephadex G-200
equilibrated with 0.05 M sodium phOSphate buffer50.001 M
EDTA-O.1 M ammonium sulfate (pH 7.5). Fractions (15
drOps) were eluted with the same solution and.collected
in a Gilson Medical Electronics linear fraction collector.
The elution pattern is shown in Fig. 17. Fractions 42 to
55, which had the highest Specific activity, were com-
bined. The 6-PFK activity in the combined fraction was
6-fOld purified over the crude extract.

.Attempts to purify the enzyme further by DEAE-
cellulose column chromatography, calcium phOSphate gel
adsorption, and pH fractionation led to a considerable
loss Of 6-PFK activity. The Sephadex fraction had con-
taminating activities Of adenylate kinase (0.25 unit/mg).
mannitol-I-P dehydrogenase (0.12 unit/mg), 1-PFK (0.06
unit/mg), and FDPase (0.27 unit/mg).

Purification Of 1-Phosphofructokinase

The prOcedure described here for the purification
Of i—PFK is a modification and extension Of the procez
-dure of Hanson.and.Anderson (54). All Operations were
performed at 0-4°C. A summary Of the purification is
given in Table IX.

Protamine Sulfate Precipitation

The protein concentration Of the crude extract

was adjusted to 8 mg per ml. Nucleic acids were precipi-

 

 

 

 

107

Figure 17: Elution pattern Of 6APFK on Sephadex
G-200 column. Details are given in

the text.

 

108

 

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poawdooam mambooom pr09 Hence mafiao> Soapowam

Himd.pamuda 809w ommzaxopodaaozmmosgla ho zoapmoamzham

NH MAm¢B

 

 

 

110
tated by the slow addition, with constant stirring, of
20% by volume of 2% aqueous solution Of protamine sul-
fate (pH 7). The precipitate Obtained by centrifugation

was discarded.
Ammonium Sulfate Fractionation

Solid (NH4)280u was added slowly with stirring
to the protamine sulfate supernatant and the precipitate
which formed between 0 and 30% saturation was removed by
centrifugation and discarded. The fraction precipitat-
ing between 30 and 60% saturation was collected by cen-

trifugation and dissolved in glass-distilled water.
Sephadex G-200 Chromatpggaphy

Sixteen m1 Of the above fraction was layered on a
column (45 x 4.5 cm) Of Sephadex G-200 equilibrated with
0.02 M sodium phOSphate buffer (pH 7.5). The same buffer
solution was used to elute 4OO-drOp fractions, which were
collected on a Gilson Medical Electronics fraction col-
lector. The elution profile is shown in Fig. 18.
Fractions 43 to 52, containing the highest 1-PFK Specific
activity, were combined.

Ca1cium.PhOSphate Ge1.Adsgrption
Mulch" * *

The combined Sephadex fraction (1.2 mg protein per

ml) was treated with 10% by volume Of calcium phOSphate

I.“

111

Figure 18: Elution pattern of 1—PFK on a Sephadex
G-200 column. Details are given in

the text.

 

112

 

 

 

 

Figure 18
(“m HHd 911m) mAImov
s o \o a «2 a
oi oi .1 .4 o eI
I I I I is
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38 .
ES /’
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E \51' __;\O
— “r P ‘n
H O
\ ..
O"
\
_ . d—s
O O \
O
O
_- . —‘g
0
O
O
__ E. . —‘3’\
[in
E-0 O
8
a. O
\\\\’ 0
. :-
—— O “N
O
O
I
\0
—— F:
I |
\O .& cu

('IN and em) NIHIOHd

FRACTION NUMBER

 

 

 

113

gel containing 62 mg solids per ml. The suSpension was
stirred well for 5 minutes and centrifuged. The gel
solids were washed with 0.02 M sodium phOSphate buffer
(pH 7.5), and treated twice (10 min per treatment) with
0.075 M sodium phOSphate buffer (pH 7.5). The superna-
tant from each treatment was discarded. 1-PFK activity
was eluted by mixing the gel solids well with 0.15 M
sodium phOSphate buffer (pH 7.5) for 15 min. The elu-
tion was repeated to recover about 80% of the adsorbed

1—PFK. The two eluates were combined.
Ammonium Sulfate Precipitatign

The combined eluate was concentrated by precipi-
tation with solid ammonium sulfate (70% saturation) and

dissolving the precipitate in glass-distilled water.
pH Fractionation

The concentrated gel eluate (10 mg protein per ml)
was diluted two-fold with glass-distilled water. The pH
Of the solution was carefully adjusted, with constant
stirring, to pH 4.6 with 7.5% acetic acid. The superna-
tant Obtained upon centrifugation was treated with 0.1 M
NaOH to pH 7.5. 1-PFK activity in this preparation was
approximately 315-fold purified over the crude extract-
and was essentially free from adenylate kinase ((0.0003

unit/mg), mannitOl-l—P dehydrogenase (<0.0007 unit/mg),

 

 

 

114
and FDPase ((0.0003 unit/mg).

Exgpegties and Regulation Of
6fiPhOSphOfructOkinase and 1-PhOSphOfructokinase

 

Stability

6-PFK readily lost activity in crude extracts pre-
pared in water, but was stable at -20°C for approximately
1-2 weeks in 0.05 M sodium phOSphate buffer-0.001 M EDTA
(pH 7.5). The Sephadex G-200 fraction.C1n 0.05 M sodium
phosphate buffer-0.001 M aura-0.1 M (NH4)ZSOu (pH 7.51]
was stable at -20°C for about two months.

lzPFK was stable for weeks at -20°C in crude
extracts prepared in water. The 315-fold purified enzyme
preparation (pH 4.6 supernatant) in 0.1 M (N34)2304 (pH
7.5) was stable for at least 8 months at -20°C if not

repeatedly thawed and frozen.
ATP Inhibition

At 0.33 mM Defructose-6-P and 2.0 mM Mg*+. 6-PFK
was inhibited by ATP at concentrations above 1.0 mM. The
inhibition became undetectable when the Mg++ to ATP ratio
was maintained at 2:1 from 0.6 mM tO 3.3 mM ATP (Fig.

19 A & B). Raising the concentration Of D-fructose-6-P
to 3.3 mM while keeping the hg++ constant at 2.0 mM also
served tO reverse the previously Observed inhibition by

ATP (Fig. 19B).

 

s“ .

Figure 19:

115

Inhibition of 6-PFK by ATP in the presence
of various concentrations of Mg++ and
D-fructose-é-P. Mg++ and.ATP were varied
as indicated in the plots. A. Effect of
ATP in the presence of 0.33 mM D-fructose-
6P. B, Effect of ATP in the presence of
3.3 mM D-fructose-6-P. The amount of

enzyme in all assays was 0.0015 unit.

 

11.6

 

 

 

 

 

 

 

 

Figure 19
(NIH/smom) cameos ads
0
(x;
T
_ N
u
“r
«3 £3:
1 t. e: f
n e presene o
, ++ (— 8 g)
of Mg and ::>
i 93.
ST? were varie'. :5
A, Effect of F E
3 mil D-fructose- “3
m
T“ A
.19 presence 0. m
3 amount 0f
0
.0015 unit. N
(— n — ‘0
“n. 3‘
\c; i
m i
L. m ‘10
8 S _ m
o
E
I
Q
L— m fl H
M
O
3 J
m to
O {\ 1.0 N
.3 o' o o
(NIN/SH’IOIAIN) GHWHOJ CHM

 

 

mM A‘I?

 

 

 

 

117

ATP at concentrations above 1.7 mM inhibited 1-PFK
when the Mg*+ and D-fructose-l-P concentrations were main-
tained at 3.3 mM and 0.27 mM, reSpectively (Fig. 20A).
As observed with 6—PFK, the inhibition was prevented by
the addition of Mg++ at twice the concentration of ATP
in the assay throughout the range of ATP concentrations
tested. However, raising the level of D-fructose-l-P
from 0.27 mM to 6.7 mM and-keeping the Mg++ constant at
3.3 mM had no effect on the inhibition of ATP at concen-
trations above 1.7 mM (Fig. 20B). I
interaction of Hexose.Pho§phate Substrate

an

A plot for 6-PFK of rate vs D-fructose-6—P concen-
tration (Fig. 21A) gave a sigmoidal curve, which became
more marked as the Mg+f concentration was decreased while
ATP was maintained at 2 mM. The increase in the sigmoidal
character of the curve at lowered Mg“*’+ concentrations
indicates a weakened affinity of the enzyme for D-fruc-
tose-6-P. As shown in the plot, the apparent Km values
for D-fructose-6—P depended on the Mg*+ to ATP ratio and
were approximately 0.3 mM at a ratio of 2:1, 0.6 mM at a
ratio of 1.021, and 1.4 mM at a ratio of 0.7:1. Increased
levels of D-fructose—6-P relieved ATP inhibition, with
more D-fructose-6-P being required at low Mg++.concentra-
tions. The shapes of the curves suggest a cooperative

interaction between Mg++ and D—fructose-6-P (103).

 

 

 

 

 

 

Figure 20:

118

Inhibition of 1-PFK by ATP in the presence
of various concentrations of Mg++ and

D—fructose—l-P. Mg++ and ATP were varied
as indicated. The amount of enzyme in all
assays was 0.0011 unit. A, Effect of ATP
in the presence of 0.27 mM D-fructose-l-P.
B, Effect of ATP in the presence of 6.7 mM

D-fructose-l—P.

 

in the presence

1“ Mg“ and

TP were varied

f enZyme in 21'.

Effect of ATP
D-fructcse-l-P.

senoe of 6.7-:1:

 

 

 

119
2 ..
Figure 0 (MIN/9310mm (RENEW “a
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m 0 m
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(NIM/ 9310M) GHMHOJ dad

 

 

 

 

Figure 21:

120

Dependence of initial velocity on the
hexose phOSphate concentration under
conditions of varying Mg++ to ATP
ratios. ATP was maintained at 2 mM
throughout the determination. Mg++
was varied as indicated. A, Plot of
rate vs D-fructose-6-P concentration
for the 6-PFK reaction. The amount of
enZyme in all assays was 0.0018 unit.
B, Plot of rate vs D-fructose-l-P con-
centration for the l-PFK reaction.

The amount of enzyme in all assays was

0.0016 unit.

city on the
tion under
toAP
datZM
om Ng+
A, Plot of
centration
he amountof
cowtmm
ase-l-P cor
aaction.

l assays ”33

 

 

 

 

 

 

 

 

121
Figure 21
' 9 . 0 UN
N H J 6
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+ +50 g?
+ a
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(NIN/SETOMN) GHMHOJ ads

 

 

 

122
On the other hand, a plot of rate vs D—fructose-
i-P concentration with l-PFK (Fig. 21B) shows a hyper-
bolic dependence of initial velocity on D-fructose-l-P
concentration under conditions of varying Mg++ and a

constant ATP level of 2 mM. The apparent Km of 1-PFK

 

remained constant at about 0.7 mM with varying amounts
of Mg++ in the assays. The absence of sigmoidicity in
the Michaelis-Menten plot for 1-PFK suggested the
inability of D-fructose-l-P at the concentrations
tested to relieve ATP inhibition.
The Km of 1-PFK for D—fructose-l-P (0.75 mM)

did not vary with ATP concentration (Fig. 22). D—Fruc-
tose-l-P concentration had no effect on the Km for ATP,

which remained constant at approximately 0.7 mM (Fig. 23).

Effect of Mg++

 

Fig. 24A shows that with 6—PFK, a sigmoidal curve
of rate vs Mg++ concentration was obtained at a low
D-fructose-6-P concentration (0.33 mM). An increase in
the D-fructose-6-P level to 1.33 mM led to a shift from
a sigmoidal to a regular hyperbolic curve, indicating
that increased D-fructose-6—P concentrations could decrease
the requirement of the reaction for Mg++.

A similar plot for 1-PFK also shows a sigmoidal
dependence of rate on the Mg++ concentration (Fig. 24B).

++
However, D—fructose-l-P could not substitute for Mg , as

 

 

 

 

 

 

123

Figure 22: Lineweaver-Burk plot for the determina-
tion of the Km of 1—PFK for D-fructose-
l-P in the presence of various ATP con-
centrations. ATP and D—fructose-l-P
were varied as indicated, and Mg++ was
maintained at twice the ATP concentra-
tion throughout the determination. Thé
amount of enzyme in all assays was 0.001

unit.

 

she determina-
;r D-fructose-
‘ious ATP con-
'uctose-l-P

and Hg“ was

31: concentra-

tination. The

:says was 0.001

Figure 22

124

 

 

K.m FOR D-FRUCTOS E-l —P

 

0.75 mM

l

0.45 mM ATP

0.67 mM ATP

 
 

 

 

 

100 -—

75—

50—-

-1

—2

 

DnFRUCTOSE-l ~P (mM)

 

 

125

Figure 23: Lineweaver-Burk plot for determining the
Km of 1-PFK for ATP in the presence of
various concentrations of D-fructose-l-P.
ATP and D—fructose-l—P were varied as
indicated, and Mg++ was maintained at
twice the ATP concentration. The amount

of enZyme in all assays was 0.001 unit.

 

 

 

126

Figure 23

 

 

 

2%: was

 

HI-

 

 

mIHImmOposhmIQ SE 5.0

 
    

 

 

 

I: mm
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O
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m. m a... m m m m
.m w ”W ”m m m mu
m m... m m m... T M
.Mw p «MW e m n... S

cm:
.W
0

 

 

 

127

Figure 2#: Dependence of initial velocity on Mg++
concentration in the presence of vari-
ous concentrations of the hexose phos-
phate substrate. ATP was maintained
at 4 mM, and Mg++ and hexose phOSphate
concentrations were varied as indicated.
A, Plot of rate of the 6-PFK reaction
vs Mg++ concentration. The amount of
enzyme in all assays was 0.0015 unit.
B, Plot of rate of the l-PFK reaction
vs Mg++ concentration. The amount of

enzyme in all assays was 0.0013 unit.

128

 

 

  
     
 
 
  

     

 

 

 

Figure 24
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(MIN/smoww) (IENHOJ cm

 

 

 

 

 

129
shown by the absence of a shift from a sigmoidal to a
hyperbolic curve when the D—fructose-i-P level was

raised from 0.6 mM to 6.7 mM.

Effect of Other Nucleoside TriphOSphates

 

6-PFK is non-Specific with regard to its phos-
phoryl donor (Table X). The purine nucleotides, GTP
and ITP, seem to be better donors than the pyrimidine
nucleotides, GTP, UTP, and TTP. The enzyme showed a
hyperbolic dependence of rate on D-fructose-6-P con-
centration with all the nucleoside triphOSphates
tested (Fig. 25A). At a Mg++ to nucleotide ratio of
0.7:1, the apparent Km for D-fructose-é-P with all the
nucleoside triphOSphates tested was approximately 0.3
mM, which is the same as that with ATP as a phOSphoryl
donor at a Mg++ to ATP ratio of 2:1.

CTP inhibited the activity when the Mg++ to an:
ratio was less than 2:1, and failed to do so when the
Mg++ was present at twice the concentration of CTP ‘
(Fig. 26 A & B). D-Fructose-6—P, when increased from
0.33 mM to 3.3 mM, failed to substitute for Mg‘H' (Fig.
26B). This is in contrast with the finding with ATP as
a phOSphoryl donor (Fig. 19 A & B), in which a high
concentration of D—fructose76-P relieved ATP inhibition
at a Mg++ to ATP ratio below 2:1. All the other nucleo-

side triphosphates gave results similar to those obtained

with CTP.

 

130

TABLE X

PhOSphoryl donor Specificity of
6-phOSphofructokinase and i-phOSphofructokinase

 

 

 

Nucleoside Relative Relative
triphOSphate 6-PFK activity' i-PFK activity
75 %’
ATP _ 100 100
ITP 93 43
GTP 85. 5 35
UTP 57 3-9
CTP 50 9.8
TTP 50 8.8

 

All nucleoside triphOSphates were tested at a con—
centration of 3.3 mM.

 

 

 

 

 

Figure 25:

131

Dependence of initial velocity on the
concentration of the hexose phOSphate
substrate with other nucleoside triphos-
phates as phOSphoryl donors. Mg++ and
nucleoside triphOSphate were maintained
at 1.33 mM and 2 mM, reSpectively, and
the hexose phOSphates were varied as
indicated. A, Plot of the rate of the
6-PFK reaction vs D—fructose—é-P concen—
tration with ITP, GTP, UTP, TTP, and C1?
as phosphoryl donors. The amount of
enzyme in all assays was 0.0015 unit.

B. Plot of rate of the i-PFK reaction
vs D-fructose—i-P concentration with GTP
and ITP as phOSphoryl donors. The amount

of enzyme in all assays was 0.022 unit.

 

:ity on the
phosphate
side triphoe
. Mg++ and
e maintained
tively, and
varied as
rate of the
e-6-P concen-
NT, and CT
amount of
3015 unit
( reaction
tion Withefi
The amount

0. 022 unit:

Figure 25
O

o
H

 

132

0.75
0.5

0.25

 

2.0 leI NUCLEOSIDE TBIPHOSPHA‘I‘E

ITP

mM D—FBUCTOSE—1_P

 

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mM DaFRUCTOSEaé—P

 

 

 

 

 

Figure 26:

133

Inhibition of 6-PFK by CT? under condi-
tions of varying D-fructose-é-P and Mg++
concentrations. GTP and Mg*+ were

varied as indicated. The amount of

enzyme in all assays was 0.0015 unit.

A, CTP inhibition in the presence of

0.33 mM D-fructose-é-P. B, CTP inhibi-
tion in the presence of 3.3 mM D-fructose-

6"P o

P under condi-
se-6-P and???+
Mg++ were

2 amountof
0.0015 unit.
presenceof

B, CTP inhibi'

3.3 mil 33-mont-

 

 

 

Figure 26

134

(mm/mom) cameos dCId

 

B) 3.3 mM D-FRUCTOSE-6-P

 

 

 

 

mMCTP

 

 

 

II +
"' e Ts 4"“
(3-0 0
I \ fl
“2 i E
m b0 M
(I) S '
n. 8 . m AN
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d I
in :3 M N H
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(NIw/ss'IowN) dswaoa EKM

 

135

GTP and ITP served as substrates for the i-PFK
reaction at 35% and h3%, reSpectively, of the rate
observed with ATP as the phOSphoryl donor (Table X).
Only a slight activity ((10% of the rate with.ATP) was
observed when ATP was replaced with CTP, TTP, or UTP.
As with.ATP, the rate showed a hyperbolic dependence
on D-fructose-i-P concentration with GTP or ITP as phos-
phoryl donor at a Ng++ to nucleotide ratio of 0.7:1
(Fig. 25B).

Effect of Nucleoside Diphosphates

At a Mg'H' to ATP ratio of 0.7:1. all of the
nucleoside diphOSphates tested at 1.0 mM shifted to the
left the sigmoidal curve for 6-PFK of rate vs D—fruc-
tose-6-P concentration (Fig. 27). The curves with ADP.
IDP, and GDP were more hyperbolic than those with GDP
and UDP: therefore the purine nucleotides seem to be
more effective in relieving ATP inhibition at low D-
fructose-6-P concentrations. None of the nucleotides
inhibited or activated the enzyme at concentrations of
D-fructose-éeP sufficient to give maximal rate.

None of the nucleoside diphOSphates (3.3 mM)
exhibited any effect on iéPFK activity when the Mg'"+
concentration was maintained at twice the total concen-
tration of the nucleotides in the assay. No relief of
ATP inhibition was observed at 2.0 mM ATP, 1.3“ mM Mg++.
and 0.2 mM D-fructose-i-P.

Figure 27:

136

Dependence of the rate of the 6-PFK
reaction on D-fructose-éqP concentra-
tion in the presence of various nucleo-
tides. ‘All the nucleotides were tested
at a concentration of 1.0 mM. Mg++ and
ATP were maintained at 1.3 mM and 2 mM,
reapectively, and D-fructose-é-P was
varied as indicated. The amount.of

enzyme in all assays was 0.0017 unit.

FDP FORMED ( NMOLES/MIN)

1.6

1.1!-

1.2

1.0

0.8

0.6

0.1+

0.2

Figure 27

137

 

7 l

1033 mm M8++

2.0 mM ATP

 

0.5

 

1.0

+1
+1
+1
+1

+1
+1

+1

1

1.5

CONTROL

 

 

mM IDP
mM GDP
mM ADP
mM AMP

mM UDP
mM CDP

mM CYC LIC AMP

J l

2.0

‘ mM D-FBUCTOS E-6-P

 

2.5

138
Effect of Nucleoside MonophOSphates

AMP was found to relieve ATP inhibition of 6-PFK
(Fig. 27). However, due to the presence of a consider-
able adenylate kinase activity in the partially purified
enzyme preparation, it is not certain whether.AMP itself
or.ADP was the compound reSponsible for reversing the
inhibition. .Atkinson and Walton (95) reported a similar
reversal of ATP inhibition in E, 22;; 6-PFK by both.ADP
and.AMP; however, it was later established (96) that,
with an adenylate kinase-free enzyme preparation, only
ADP was effective. Therefore, the previously observed
effect of AMP was actually an artifact due to the conver-
sion of AMP to ADP catalyzed by the adenylate kinase con-
taminant in the partially purified g, 221i,preparation.

The effect of other nucleoside monophOSphates
(cyclic AMP, CMP, CMP, IMP, and UMP) on 6-PFK was tested
under different conditions. .At inhibiting ATP levels
(0A mM D-fructose-6-P, 1.3 mM Mg‘H'. and 2.0 mM ATP),
none of the above nucleotides, when tested at 3.3 mM,
released.ATP inhibition. With a non-inhibiting ATP
level of 0.67 mM, neither inhibition nor activation was
detected.

None of the above nucleoside monOphOSphates,
when tested at 3.3 mM, had any effect on i-PFK activity
when the Mg++ concentration was maintained at twice the

total concentration of the nucleotides in the assay. A

 

139
slight apparent enhancement of ATP inhibition was detected

under conditions of inhibiting ATP level (1.3 mM Mg'H',

2.0 mM ATP, and 0.2 mM D-fructose-lAP). This effect was
likely due to the unavailability of some of the Mg++ for
the formation of MgATP complex since Mg++ can also form

a complex with other nucleotides.
Substrate Specificity

The non—Specific pyruvate kinase-lactate dehydro-
genase-linked assay for léPFK was used to test for pos-
sible phOSphorylation of D-fructose and several sugar
phOSphates other than D-fructose-iaP. With 0.0056 unit

of 1-PFK, no phOSphorylation «0.03% of the rate with

 

 

D-fructose-l-P) was observed with the following compoundsi,
D-fructose, L-fructose—iéP, D-mannose-6-P, D-fructose-6-P,
D-glucose-6—P, and D-glucose-ieP. L-Fructose-i-P was
tested at a concentration of 10 mM, while the rest were
tested at 33.4 mM.
The specific aldolase-linked assay was used to
test for inhibition of phOSphorylation of 0.53 mM D-
fructose—1-P. The following compounds, when added at 33.4
mM, did not inhibit: D—mannose-6-P, D-glucose-6-P, D-
glucose-iAP, and D-fructose. No inhibition was observed
with 10 mM L-fructose-i-P. A concentration of 33.4 mM
D-fructose-6-P, however, totally inhibited the reaction.
The 64PFK preparation had not been purified
enough to permit meaningful substrate Specificity studies

to be conducted.

 

 

 

 

 

140
Test.for_lnhibition of 6-PFK by
D-FructoseiLfl:

Mutant DD31, lacking 1-PFK activity, was employed
to test for possible inhibition of D-fructose-ééP phos—
phorylation by D-fructose-l-P. The mutant was grown on
D-glucose, and 6-PFK was purified 6-fold as described
for the wild type. D-Fructose-l-P at 6.7 mM did not
inhibit the phOSphorylation of 0.33 mM D-fructose-64P
in an aldolase-linked assay.

Inhibition of i-PFK by
D-Fructose-E—P

The Specific aldolase-linked assay was used to
test for inhibition of the phOSphorylation of D-fructose-
14F by D-fructose—6-P. The inhibition was competitive
with D—fructose-l-P (Fig.’28) and the Ki for D-fructoseu
64F, as determined from a kinetic plot (Fig. 29), is

approximately 1.5 mM.
FDP Inhibition

The pyruvate kinase-lactate dehydrogenase—linked
assay for 1-PFK was used for the inhibition study with
FDP. .As shown in Fig. 30, FDP competitively inhibited
D-fructose—iaP phOSphorylation. The Ki for FDP was
approximately 7 mM (Fig. 31).

The presence of FDPase contaminant in the 6+PFK

preparation precluded the determination of the effect of

FDP on 64PFK activity.

 

Figure 28:

141

Lineweaver-Burk plot showing the rela-
tionship of D-fructose-léP concentra-
tion to 1-PFK reaction velocity in the
presence of various concentrations of
D-fructose-64P. The routine aldolase-
linked assay was used, except that D-
fructose-l-P and D—fructose-6-P were
varied as indicated. The amount of

enzyme in all assays was 0.001 unit.

142

Figure 28

25 e. Tm moaoomaua

 

a
H

N-

 

I

II

 

mlolmwoaoDMMIQ
as o

AlwlmmOBODmhIQ
2E H

O

mlwlmmoeobmmlm
SEN

N
fl

_

 

 

 

mm

on

3.

God

93le

 

 

 

 

Figure 29:

143

Kinetic plot for the determination of
the K1 of 1—PFK for D-fructose—6-P.
The data are taken from the eXperi-

ment described in Figure 28.

 

144

Figure 29

 

 

mIQIMmOEDDmmIQ 2E

NI.

 

IlmlalmmOBODmmIQ
SE o.N

mIHImmoaosmaIs
as w.o
muaummoaosmanm
as no.0

mIHImmoeopmmIm
as mm.o

TI

 

 

 

SE m.a H mlwIHWOBODmmiQ

 

mom Ha

ll ooa

 

 

 

 

Figure 30:

145

Lineweaver-Burk plot showing the rela-

tionship of D-fructose-l-P concentration
to l-PFK reaction velocity in the
presence of various concentrations of
FDP. The routine pyruvate kinase-lactate
dehydrogenase-linked assay was used,
-except that D-fructose-l-P and FDP were
varied as indicated in the plot. The
amount of enzyme in all assays was 0.001

unit.

 

146

Figure 30

m

Aasv mIHImmoeosmaIn

 

 

mmmzfio

.maa as m.m

mgm 25 5.0

O

1

 

 

 

 

mm

on

ooa

 

Figure 31:

147

Kinetic plot for the determination of
the K1 of l—PFK for FDP. The data are

taken from the experiment described in

Figure 30.

 

 

 

o e:

 

148

Figure 31

 

mIHIMWOEODmmID :8 o.N

mIHImwOBODmmIQ SS m.o

asanamoaoamaIm as mm.o

mIH

ImmOBoDmmIQ SS mm.o

as w.o

 

n mam mos Ha

 

 

mm

om

OOH

 

149
Effect Of P1

P1 at 2.6 mM did not relieve ATP inhibition of
6-PFK when the Mgf"+ to ATP ratio was 0.7:1; neither
inhibition nor activation could be detected when the
Mg++ concentration was twice the.ATP concentration.

Pi had no effect also on 1-PFK activity when
tested at 2.7 mM under conditions of inhibiting or non-

inhibiting ATP levels.
Effect of_citrate

Citrate at 6.7 mM had no effect on 6-PFK activ-
ity under conditions of limiting ATP and D-fructose-6-P
(0.67 mM ATP, 0.67 mM D-fructose-6-P, and 1.3 mM Mg++).

In contrast, citrate was found to be a competi-
tive inhibitor of D-fructose-l-P in the l-PFK reaction

(Fig. 32). The Ki for citrate was approximately 0.85

mM (Fig. 33).
grrect of on

.A pH-activity profile of 6-PFK (Fig. 34A) gave a
broad curve, with Optimum activity at about pH 8. No
deviation from sigmoidal kinetics was observed over a
pH range from pH 7 to pH 9 (Fig. 35). There was no
apparent increase in.ATP inhibition at low pH values,
and the effectivity of D—fructose-6-P in relieving ATP
inhibition did not seem to be affected by the pH of the

reaction.

 

 

 

Figure 32:

150

Lineweaver-Burk plot showing the rela-
tionship of D-fructose-i-P concentration
to 1+PFK reaction velocity in the pres-
ence of various concentrations of citrate.
The routine aldolase-linked assay was
used, except that D-fructose-i-P and
citrate were varied as indicated. The
amount of enzyme in all assays was

0.0018 unit.

151

aasv mIHIamoaoaaaus

H
0

HI!

N-

 

 

Figure 32

MBQmBHU SE C

mafimaHo ES mm.o

mafimeHo SE o.H

 

Ime

 

 

 

Figure 33:

152

Kinetic plot for the determination of
the K1 of 1-PFK for citrate. The data

are taken from the eXperiment described

in Figure 32.

 

 

153

Figure 33

m.H o.H

HB<mBHU 28

m6

 

 

 

\o
mIHImmOBUDmmIQ ES o.N

mlHIMWOBUDmmID ZS o.H

mlHlmmOBODmle 25 0.0

mIHImmOEoDmmIQ SE :.0

:8 mm.o N m94meHo mom

_

 

.H
La:

.lom

 

 

O

Figure 34:

154

pH-activity profiles of 6-PFK and 1-PFK.
The buffers (0.067 M) used were cacodylate,
pH 6.0 to 7.0, glycylglycine, pH 7.0 to
8.0, and glycine, pH 8.5 and 9.0. pH
measurements were made in duplicate reac-
tion mixtures. A, pH-activity profile of
6-PFK. B, pH-activity profile of iaPFK.

155

Figure 34

(\-

mm

 

 

_.J\o

 

azH owns I
mfiofiofloflo ouo

magma 040 DID

MmmIH Am

_ _

—

Mmme A4

0N

0.:

ow

om

OOH

 

 

KELIAIELOV HAI IIIV'IHH

 

156

Figure 35: Dependence of rate of the 6—PFK reac-
tion on D—fructose-6—P concentration
at different pH values. The buffers
(0.067 M) used were glycylglycine, pH
7.0 to 8.0, and glycine, pH 8.5. ATP
and Mg++ were maintained at 2 mM and

2.7 mM, reSpectively.

 

157

Figure 35

 

 

 

T

 

2.7 mM Mg++

2.0 mM ATP

 

 

 

 

2.0

1.5

mM D-FBUCTOSE-6-P

0.5

 

 

 

HBH>HBU<Vm>HBdem

158
A pH-activity profile of 1-PFK (Fig. 348) showed
that the pH Optimum of the reaction is pH 7.5. No shift
in the pH curve was observed when the D-fructose-l-P
concentration was varied in the range of 0.2 mM to 6.7

mM.

Molecular Weight Determination

The molecular weights of 6-PFK and 1-PFK were
estimated by Sephadex G-100 chromatography as described
by P. Andrews (104). Pig heart malic dehydrogenase
(MW 70,000) (105), yeast alcohol dehydrogenase (MW
150,000) (106, 107), and horse heart cytochrome c
(MW 12,400) (108) were used as molecular weight standards.
Sodium phosphate buffer (0.02 M, pH 7.5) was used to
equilibrate the column (25 x 1.2 cm) of Sephadex G-100
and to elute 20-drop fractions. The elution pattern of
the proteins is shown in Fig. 36. From a plot of elu-
tion volume, Ve, versus log MW of the standards (Fig. 37).
the molecular weight of 6—PFK was estimated to be approx-
imately 100,000 and that of i-PFK to be approximately
75,000.

 

Figure 36:

Elution profile of 1—PFK, 6-PFK, malic
dehydrogenase, alcohol dehydrogenase,
and cytochrome c on a Sephadex G-100
column. Details are as described in

the text.

 

160

099,

Figure 36

.0“

mmmzsz ZOHBUE

on mm on

 

   
 

  

 

ma mm om
. _. _ i . _
5 mmfifioomfimas . .f
Basal. \. 2 . / ammo
H00 III C C. 0 II
o maomaooeao.\\x . . unease
N6 Iowans
I . .8834 I
ass...“ \ .

 

 

 

ILIAIIIOV 3A1 ILV'IHH

 

161

Figure 3?: Plot of elution volume Ve vs log MW of
the standards for the estimation of the

molecular weights of 1-PFK and 6-PFK.

 

 

 

162

Figure 37

 

ON

mm

on

mm

on

UN
:3‘

 

 

 

:IOH N BmUHmz m¢HDOMHOE

 

 

 

     
     

 

 

om 3 3.0 m s c m a m H
_. H _ _ _ _ n _ _ _ _
_ i om
. N H
mmaaaooamaaani\. ooo maaamfi
qoaooqs .\
08.03 H 3a J mm
ammuc
mmaaaoomaaaaa Beta \.
.L Om
m
>
J on
o aaomaoosao I.\.
_ _ _ .[n _ _ _

 

 

 

 

DISCUSSION

6-PFK functions in D-glucose metabolism of A,
aerogenes presumably in a manner similar to that of
analogous 6-PFKs from a variety of sources. A con-
stitutive FDPase hydrolyzes the product back to D-
fructose-6-P. For this reason, the D-fructose-6-P-FDP
cycle may function as a net ATPase if not controlled
(57). If properly controlled, the cycle can regulate
both glycolytic and gluconeogenic rates and maintain
the delicate balance of nucleotides within the cell.

It is therefore necessary for the 6-PFK reaction to be
regulated. 0n the other hand, the inducible 1-PFK,

which functions in D-fructose metabolism of A, aerogenes.
is not known to participate in such a "futile" cycle.

It is therefore highly probable that i-PFK is not regu-
lated by the mechanisms governing 6-PFK activity. The
present studies were conducted to characterize 1—PFK

and to compare its preperties and control mechanisms
with those of 6-PFK from the same organism. Since 6-PFKs
from a number of organisms have already been purified
and characterized, an extensive purification of the
enzyme from.é, aerggenes was not attempted.

The findings described in this investigation are

consistent with the central role played by 6APFK in

163

164
glycolytic control and in maintaining the delicate balance
of nucleotides within the cell. More important, this
thesis establishes that 1-PFK has kinetic properties very
different from those of 6-PFK.

ATP inhibits both enzymes when the Mg++ to ATP ratio
in the assay falls below 2:1. The reaction rate with
6-PFK, like that of the enzyme from E. ggli (95), exhibits
a sigmoidal dependence on D-fructose-6-P concentration
which becomes more pronounced as the Mg++ to ATP ratio is
decreased. The apparent Km for D-fructose-6-P is depen-
dent on the relative amounts of Mg++ and ATP in the assay.
0n the other hand, 1-PFK exhibits hyperbolic kinetics with
reSpect to the D-fructose-i-P concentration even under
conditions of inhibiting ATP levels, and the apparent Km
for D-fructose-ieP remains constant with varying.ATP and

Mg++

concentrations.

A sigmoidal curve of rate vs Mg++ concentration is
obtained with both enzymes. Increased concentrations of
D-fructose-6-P shift the curve of the 6-PFK reaction from
a sigmoidal to a hyperbolic one, whereas, in the 1-PFK
reaction, increased D-fructose-i-P levels have no effect
on the sigmoidal character of the curve or on the apparent
Km for Mg“. Since Mg++ and ATP form a MgATP complex,
which is the real substrate of the reaction (113), it is
not certain whether the relief of ATP inhibition by Mg++
in the 1-PFK reaction is due to the formation of the com-

plex or to the binding of Mg*+ to a separate site.

 

 

n .
3
A}
u’\‘
r.” , 1..
. ‘i .
-.~
.A J!
I'V

 

165
Neither enzyme is strictly Specific for ATP as

phosphoryl donor. 6-PFK utilizes ITP, GTP, CTP, UTP,
and TTP, while l—PFK utilizes GTP and ITP. The mechan-
ism of inhibition of the nucleoside triphOSphates seem
to be Similar for both enZymes. In this reSpect A,
aerogengs 6-PFK behaves differently from those from
mammalian sources, where inhibition by other nucleoside
triphOSphates exhibits a pattern Similar to ATP inhibi-
tion (71, 73, 78). In yeast (92-94) and E. 321; (95),
other nucleoside triphOSphates either inhibit the activ~
ity very slightly or not at all even at low Mg++ concen-
trations.

Both AMP and ADP were found to relieve ATP inhibi-
tion of 6-PFK, but Since a considerable amount of adenylate
kinase activity was present as a contaminant in the par-
tially purified preparation, it is not clear whether both
compounds are in fact effective. A similar relief of ATP
inhibition by other nucleoside diphOSphates and the
absence of any effect with other nucleoside monOphos-
phates suggest that ADP, rather than AMP, is the active
compound. The absence of any effect on 6-PFK with cyclic
AMP and other nucleoside monophOSphates is in agreement
with the results obtained with the enzyme from yeast
(92694) and E, 33;; (95. 97). Mammalian 6-PFKs are
generally not affected by most nucleoside monOphOSphates
(71, 73, 78); cyclic AMP, however, is known to be very
effective in relieving ATP inhibition (71—75, 78, 81, 84).

 

 

166

ADP, AMP, cyclic AMP, and various other nucleoside
mono- and diphOSphateS have no effect on the l-PFK reac-
tion when the Mg++ to total nucleotide ratio in the assay
is maintained at 2:1. In some cases, a Slight inhibition
is observed at lower ratios; this effect may be due to
ATP alone, rather than to enhancement of ATP inhibition
by the other nucleotides.

pH has no effect on the extent of ATP inhibition
of 6-PFK or on the sigmoidicity of the curve of rate vs
D-fructose-6-P concentration. This finding is in con-
trast with those for several mammalian 64PFKS (72, 73, 75,
84, 99, 100). To my knowledge, no studies have as yet
been reported on the effect of pH on 6-PFKs from yeast
and other bacteria.

According to Monod, Wyman, and Changeaux (103),
allosteric proteins may be classified into either the K
system or the V system. In the K system, both substrate
and effector have differential affinities for the two
states of the protein, and the presence of one will
modify the apparent affinity of the protein for the
other. In the V system, the substrate has the same
affinity for the two states, while the effector has
differential affinities. The two states of the protein
differ in their catalytic activity. The effector will
-therefore act as an inhibitor if it has maximum affinity
for the inactive state, or as an activator if it has

maX1mum affinity for the active state. The effect of

‘

 

 

 

 

 

 

167
Mg++ and various nucleoside diphOSphates on the Km of 64PFK

for D-fructose-6-P and the constancy of the maximal
velocity suggest that A, aerogenes 6-PFK belongs to the K
system type of allosteric proteins.

,Aside from A, aerogenes 1-PFK, Dictyostelium
discoideum 6-PFK is the only PFK known to deviate from
the general regulatory pattern of a typical 6-PFK (91).
Previous investigations on this organism have revealed
that proteins and amino acids are the primary energy

sources for growth (109-111), and that the main role of

 

D-glucose seems to be to supply hexose units for cell wall
synthesis (112). It was suggested, therefore, that the
unusual regulatory pattern of the 6-PFK from this organism
may reflect an altered physiological function, that is, as
an enzyme in a supplementary energy-yielding metabolism
under conditions of high glycogen and excess D—glucose
(91).

Although A. aerogenes i-PFK is not controlled by
mechanisms similar to those operative for 6-PFK, the

observed.lg vitro inhibition of the former enzyme by

 

D-fructose-6-P, citrate, and, to some extent, FDP, may
suggest possible in zgzg control. Citrate, which is an
intermediate in the tricarboxylic acid cycle, is a feed—
back inhibitor of the enzyme. Although the inhibition
seems to be competitive with D-fructose-i-P, it is diffi~
cult to visualize citrate as binding to the D-fructose-l-P

site, since its structural formula is very different from

   

 

 

168
that of D-fructose-iAP. Further studies are needed to
elucidate the real mechanism of citrate inhibition. FDP
inhibits competitively with D-fructose-leP, but the
rather high K1 of approximately 7 mM suggests that the
inhibition may not be of major physiological significance.
D-Fructose-6-P, the product of the FDPase reaction, is a
more potent (K1 = 1.5 mM) inhibitor of 1APFK activity.

A, aerogenes 6-PFK, like that from g. ggli, was
not inhibited by citrate. 6-PFKs from various organisms
are known to be inhibited by citrate (70, 80-84, 88, 89).
The inhibition of Sheep brain (80) and rat heart 6-PFKS
(83) is competitive with D-fructose-6-P.

It is not known at this time whether the l-PFK
reaction is a rate-controlling step in D-fructose metabo-
lism of A, aerogenes. But if it is, its control mechan-
isms are very different from those of 6-PFK from the same
organism.

The Km of A, aerogenes 1-PFK for either substrate
is not affected by the concentration of the other sub-
strate. Such kinetics are consistent with a sequential
mechanism of substrate binding (41). Similar findings
have been reported for Sheep brain 6-PFK at pH 8 (71).

In contrast, Dictyogtelium discoideum 6-PFK (91) exhibits
parallel kinetics characteristic of the so-called ping-

pong mechanism of substrate binding (41).

 

 

 

 

SUMMARY OF PART II

The relative significance of the D-fructose-i-P
and D-fructose-6-P pathways in D-fructose metabolism of
A, aerogenes PBL-B3 was assessed by mutant analysis.
Mutant.A9-1, which grew well on both D-fructose and
glycerol but not on D-glucose, lacked 6-PFK activity
but showed normal levels of 1-PFK and FDPase activities.
0n the other hand, mutant 012, which grew well on D-glu-
cose but not on D-fructose or glycerol, lacked FDPase
but had a normal level of 6-PFK activity. The data thus
indicate that the pathway of D-fructose metabolism is
primarily through Dbfructose-i-P, and that the D-fructose-
6-P pathway is operational in D-glucose metabolism.

Comparative studies on the properties and regula-
tion of partially purified.6aPFK and 1-PFK from,§.
aerogenes were conducted. {A plot of rate vs substrate
concentration revealed that 6HPFK exhibits a sigmoidal
dependence of rate on D-fructose-6-P concentration,
whereas 1ePFK shows a hyperbolic dependence of rate on
D-fructose-l-P concentration. ATP inhibited both enzymes
under conditions of Mg++ to ATP ratios below 2:1. The
inhibition of 6-PFK was relieved by Mg++, D-fructose-64P,
ADP, and various other nucleoside diphOSphates. In con-

trast, the inhibition of 1-PFK could be relieved by Mg‘“+

169

170
only. Both enzymes showed a sigmoidal dependence of rate
on Mg++ concentration. Increased levels of D-fructose-6—P
shifted the 6-PFK curve from sigmoidal to hyperbolic,
whereas D-fructose-i-P had no effect on a similar plot for
1-PFK. Other nucleoside triphOSphates were used as phosu
phoryl donors by both enzymes, and inhibited activity
when the Mg++ to ATP ratio was below 2:1. In contrast
with the finding with ATP, the inhibition of 6-PFK by
other nucleoside triphOSphates was not relieved by D-
fructose-6qP, and the rate of the reaction exhibited
hyperbolic dependence on D-fructose-6-P concentration.
Citrate, FDP, and D-fructose-6HP inhibited the 1-PFK reac-
tion competitively with D-fructose-i-P, suggesting possible
igflzizg control of activity. The data indicated that
whereas 6-PFK exhibits allosteric properties and a regu-
latory pattern typical of 6-PFKs from a variety of organ-
isms, 1-PFK behaves more like a non-allosteric kinase.
The two enzymes were further characterized with reSpect
to substrate Specificity, pH optimum, and molecular
weight. The Km of 1-PFK for either substrate did not
vary with the concentration of the other. This finding
is consistent with a sequential mechanism of substrate

binding to the enzyme.

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