METABOLISM OF L-MANNOSZE
IN AEROBACTER AEROGENES

Thesis {or “to Dogma of m. D.
MCEEGAN STATE UMVEZRSETY

Joseph William Mayo
1968

 

0-169

Date

,4!
LI 23 “AF

h‘iCEl 1‘13“ Sin

Us“ diversity

.1“

 

This is to certify that the
thesis entitled

METABOLISM OF L-MANNOSE
IN AEROBACTER AEROGENES

presented by

Joseph W. Mayo

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biochemistry

fl . “a

Major professor

8/15/68

 

i

.-_.. -- -—-_

ABSTRACT

METABOLISM OF L-MANNOSE
IN AEROBACTER AEROGENES

by Joseph W. Mayo

A mutant strain of Aerobacter aerogenes PRL-Rj
was isolated wnicn, unlike the parent strain, grew
readily on the unnatural hexoses, L-mannose and L-fr’uc—
tose, as sole carbon sources. The pathway bywhich
L-mannose is degraded in this organism was elucidated.

An isomerase catalyzes the conversion of L-mannose to
L-fructose, which is phOSphorylated with adenosine 5'-
triphosphate by a kinase to yield L-fructose l-phosphate.
L—Fructose l-phosphate is cleaved by an aldolase to yield
dihydroxyacetone phosphate and L-glyceraldehyde; The two
intermediates in the pathway, L-fructose and L-fructose
l-phosphate, were isolated and characterized.

The enzymes which degraded L-mannose also degraded
the naturally occurring b-deoxy hexose, L-rhamnose; Both
L-mannose and L-rhamnose induced all three enzymes in both
the wild-type and the Lemannose-positive cells: the ratio
of the specific activities of the enzymes on L-mannose
and L-rhamnose and their respeCtive metabolic intermediates

were the same in cells grown on either substrate. L-Mannose

Joseph W. Mayo

was isomerized at a rate 25% that of L-rhamnose, L—
fructose was phosphorylated at a rate 10% greater than
L-rhamnulose, and L-fructose-l-P was cleaved at a rate
25-30% that of L-rhamnulose-l-P. Partial fractionation
of the enzymes with ammonium sulfate and Sephadex G-100
failed to separate the enzymes of the L-mannose pathway
from those of the L-rhamnose pathway. When the two sub-
strates for each enzyme were mixed, the individual
activities were competitive rather than additive. A
mutant of A, aerogenes PRL-R3 deficient in the isomerase
was unable to isomerize either L-mannose or L-rhamnose.
'The mechanism by which A, aerogenes gains the ability
to metabolize many of the unnatural hexoses, pentoses,
and penitols has been shown by other workers to involve
the selection of derepressed mutants containing’higher
levels of non-specific enzymes which have naturally
occurring compounds as their normal substrates. In
contrast to this, the gain in the ability of this organism
to grow on L-mannose as a sole carbon source did not involve
selection of derepressed mutants. L-Mannose inhibited
the growth of both the wild type and the mutant at the
onset of L-mannose utilization by the cells; only the
mutant overcame this inhibition. When the inducer, L-

mannose, was removed from actively growing mutant cells,

Joseph W. Mayo

a rapid decrease in the activities of all three enzymes
occurred. Also, L-mannose was not utilized by the mutant
cells in the presence of either chloramphenicol or pure-
mycin, further indicating that growth on L-mannose required

the induction of these enzymes.

METABOLISM OF L-MANNOSE

IN AEROBACTER AEROGENES

BY

Joseph William Mayo

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1968

Dedicated
to my

parents

ACKNOWLEDGMENT

The author wishes to express his gratitude to
Dr. Richard L. Anderson for his advice and helpful
criticisms throughout the course of this work. The
discussions and assistance of his fellow graduate
students also are deeply appreciated. Thanks are
due to Mrs, Barbara Palmer and Mrs. Shirley Randall
for their help in preparing this manuscript. The
financial support of the National Institutes of

Health also is appreciated.

ii

TABLE OF CONTENTS

ACKNOWLEDGMENTS............

LIST OF TABLES o g o o o o o o 9' o o 0

LIST OF FIGUES O O O O O Q 0 O O O C 0

INTRODUCTION .. .

PART I.

O
Q
0
C

THE BIODEGRADATION OF L-MANNOSE BY
AEROBACTER AEROGENES. . . . . . .

Experimental Procedures. . . . . .
Results . . . . . . . . . . .

Apparent phosphorylation of L-mannose

Manometric: L-mannose-stimulated
C02 evolution from bicarbonate
in the presence of ATP . . .
Titrametric: increased rate of
acid production from ATP in
the presence of L-mannose . .
Colorimetric: ATP-stimulated dis-
appearance of L-mannose . . .
Spectrophotometric test for L-
mannokinase. . . . . . .

Isomerization of L-mannose to L-
fructose. . . . . . . .
Enzymic preparation of L- fructose .
Growth of.§. aerggenes on L- fructose
Phosphorylation of L-fructose to

L— fructose- l- P. . . .
Enzymic preparation of L- fructose 1-
phosphate . . . . . . .
Identification of L- fructose 1-
phosphate . . . .
Enzymic cleavage of L—fructose 1-
phosphate . .
Reduction of L-glyceraldehyde to
glycerol. . .

Oxidation of L-glyceraldehyde to.
glyceric acid . . . .g . . .

iii

Page
ii
vi

vii

11

ll

12
12
12
21
21
33
33
36
37
‘ 41
47
47

PART II.

PART III.

Page
Discussion . . . . . . , , , 48

COMMON IDENTITY OF THE ENZYMES OF L-

MANNOSE AND L-RHAMNOSE METABOLISM. . 56
Experimental Procedures . . . . . 55
Results . . . . . . . . . . 62
Whole cell fermentation . . . . 62
Induction of enzymes by L—mannose r~
and L-rhamnose . . . 65

Properties of L-mannose (L-rhamnose)

isomerase. . . . . . . 65
Activity in crude extracts . . 65
Partial fractionation. . . . 65
Substrate specificity. . . . 77
Metal*ion actiyation: effect of
Mn and Co . . . . 7
Effect of mixing substrates. . 1
An isomeraseless Mutant (B- 22). 81
PH Optimum . . . . . . . 87
Temperature optimum . . 87
Properties of L-fructose (L-rhamnulose)
kinase. , . . . . . . . 92
Activity in crude extracts . . 92
Partial purification of the
kinase. . . 92
Km of L-fructose (L-rhamnulose)
kinase. . . . . . . 92
Substrate specificity. . . . 92
Effect of mixing substrates. . 92
pH optimum . . 104
Properties of L- fructose- l- P (L-
rhamnulose- l- P) aldolase. . . 104
Activity in crude extracts . . 104
Partial purification of the is
aldolase . . . . , , 104
Km of the aldolase. . . . . 104
Effect of mixing substrates. . 104
Substrate specificity. . . . 116
Discussion . . . . . . . . . 116
COMPARISON OF THE UTILIZATION OF L-
MANNOSE BY WILD-TYPE A. AEROGENES
AND THE L-MANNOSE-POSITIVE MUTANT. . 122
Experimental Procedures . . . . . 122

iv

REFERENCES

Results. . .

Growth of wild-type cells on D-
glucose, L- -rhamnose, and L-
mannose. . . . . . . .

Selection of the L—mannose-positive
mutant . . . . . . . .

Stability of the L-mannose-positive
mutant . . .

Response of the wild type and mutant
to Imvic tests and a specific
phage. . . . . . . .

Diauxie curves--effect of L-mannose
on the growth of the wild type
and mutant. . . . . . .

Hexose utilization as a function of
growth . . . . .

Induction of L-mannose degradative
enzymes in the wild type . .

Measurement of enzymatic activity
upon removal of the inducer .

Effect of protein inhibitors on
the utilization of L-mannose
and L-rhamnose . . . . .

Contribution of glyceric acid to
L-glyceraldehyde metabolism .

Effect of glycerol on the growth
of the wild-type and mutant
cells . . . . . . . .

Effect of DL-glyceraldehyde on the
growth of the wild-type and
mutant cells . . . . . .

Growth of the wild type and mutant
on L-galactose . . . . .

Discussion . . . . . . . .

Summary . . . . . . . . .

Page

123

123
123
126

126

126
141
148
148

154
159

159

160
160
166
172
174

Table
I.

II.

III.

IV.

VI.

VII.

VIII.

IX.

XI.

XII.

XIII.

XIV.

LIST OF TABLES

Water content of L—fructose . . . . . .

Phosphate to fructose ratio of L-fructose-l

-Po 0 o o o o o o o o o o 0

Induction of L-mannose and L-rhamnose enzymes
in Aerobacter aerogenes . . . . . .

Substrate specificity of the isomerase . .

Effect of various metal ions on the isomeriza-
tion of L-mannose . . . . . . . .

Effect of various metal ions on the isomeriza-

tion of L-rhamnose . . . . . . . .

Effect of Mn++ and Co++ on the isomerization
of L-mannose and L-rhamnose . . . . .

Effect of mixing substrates on isomerase
aCtj-Vity. o o o o o o o o o o 0

Enzyme activities in extracts of wild type
(PRL-Rj) and a L-mannose/L-rhamnose-
negative mutant . . . . . . . . .

Partial purification of L-fructose (L-
rhamnulose) kinase from wild-type cells .

Substrate specificity of the kinase . . .

Effect of mixing substrates on kinase
activity . . . . . . . . . . .

Partial purification of L-fructose-l-P
(L-rhamnulose-l-P) aldolase from the
L—mannose-positive mutant. . . .

Effect of mixing substrates on aldolase
activity. . . . . . . . . . . .

Substrate specificity of the aldolase. . .

vi

Page

.32
38

70
78

79.
80
84

85

86

95
102

105

111

‘ 117
118

Table

XVII.

XVIII.

XIX.

Page

Response of the wild type and mutant

to Imvic tests . . . . . . . . 131
Induction of L-mannose enzymes in wild-

type cells . . . . . . . . . 151
Determining the number of generations

upon removal of the inducer. . . . 152
Measurement of enzyme activity upon

removal of the inducer . . . . . 15}

Induction of L-glyceraldehyde reduc-
tase o o O o o o o o o o o 163

LIST OF FIGURES

 

 

Figure
1. Synthesis of L-mannose from L-arabinose . . .
2. L-Mannose stimulated C02 evolution from bicar-
bonate in the presence of ATP . . . . . .
3. Titrametric measurement of acid production from
ATP in the presence of L-mannose. . . . .
4. ATP-stimulated disappearance of L-mannose . .
5. Lactate-dehydrogenase-pyruvate kinase-linked
assay for the apparent phosphorylation of
L-mannose with ATP . . . . . . ._ . .
6. Chromatography of the products of L-mannose
isomerization . . . . . . . . . . .
7. Equilibrium of the L-mannose 2i L-fructose
interconversion . . . . . . . . . .
8. Crystals of L-fructose. . . . . . . . .
9. Enzymatic preparation of L-fructose--genera1
outline. . . . . . . . . . . . .
10. Growth of A, aerogenes PRL-Rj on L-fructose. .
ll. Enzymatic preparation of L-fructose-l-P--
general outline . . . . . . . . . .
l2. Acid hydrolysis of D-fructose-l-P, D-fructose—
6-P, and the product of L-fructose phos-
phorylation . . . . . . . . . . . .
13. Gas chromatography: preparation of the tri-
methylsilyl derivatives-of fructose-l-P . .
14. Triose formation from L-fructose-l-P . . . .
15. Pathway of L-mannose degradation in Aerobacter
aerogenes PRL-R3 . . . . . . . . . .
16. Equilibrium of the L-rhamnose :! L-rhamnulose

interconversion . . . . . . . . . .

vii

Page
10

14
16
18
2O
23

25
29

30
35

37a

40

43
45

60

Figure Page

17. Fractionation of L-rhamnulose-l-P on

Dowex-l-formate . . . . . . . . . . 64
18. ‘Whole cell fermentation. . . . . . . , 67
19. Whole cell fermentation of D-glucose, L-

mannose, and L-rhamnose . . . . . . . 69
20. Activity of L-mannose (L-rhamnose) isomerase

in crude extract . . . . . . . . . . 72
21. Fractionation of the isomerase on Sephadex

(5-100 0 e o e o o e o e e e o e 7).}
22. Fractionation of the isomerase on DEAE-

Sephadex o o e e e e e e e o o o 76

. ++ ++

23. Effect of varying the Mn and Co concen-

trations on isomerase activity . . . . . 83
24. pH optimum of the isomerase . . . . . . 89
25. Temperature optimum of the isomerase . . . 91

26. Activity of L—fructose (L-rhamnulose) kinase
in crude extracts . . . . . . . . . 94

27. Fractionation of the kinase on Sephadex
6.100 a Q o o e o e e e e o o e 97

28. Lineweaver-Burk plot of L—fructose (L-
rhamnulose) kinase from L-mannose-positive
cells 0 o e o O o o o o e o e e 99

29. Lineweaver-Burk plot of L-fructose (L-
rhamnulose) kinase from wild-type cells . . 101

30. pH optimum of the kinase in wild-type cells . 106

31. pH optimum of the kinase in the L-mannose-
positive cells . . . . . . . . . . 108

32. Activity of L-fructose-l-P (L-rhamnulose-l-P)
aldolase in crude extracts . . . . . . 110

33. Fractionation of the aldolase on Sephadex
G-lOO e e e o o o o e e e o o o 113

viii

Figure
34.

35.

36.

37.
38.
39.
40.

41.

42.

43.

an.

45.

46.

47.

Lineweaver-Burk plot relating aldolase
reaction velocity to substrate concen-
tration O O C O O O O O O O O

Biodegradative pathways of L-mannose and L-

rhamnose in A, aerggenes PRL-R3 . . .

Growth of wild-type cells on D-glucose, L-
rhamnose, and L-mannose . . . . . .

Selection of the L-mannose-positive mutant.

Stability of the L-mannose-positive mutant.

Phage specificity of A, aerogenes . . .

Growth of wild-type A, aerogenes on D-glucose,

and mixtures of D-glucose and L-rhamnose,
and D-glucose and L—mannose . . . . .

Growth of the L-mannose-positive strain on
D-glucose and mixtures of D—glucose and
L-rhamnose, and D-glucose and L-mannose .

Growth of the wild-type cells on L-rhamnose,
L-mannose, and a mixture of these L—hexoses .

Growth of the L-mannose-positive strain on L-
rhamnose, L-mannose, and a mixture of these

hexoses . . . . . . . . . . .

Determination of L—mannose and L-rhamnose
utilization by actively growing wild-type
cells C O O O O O O O O O O O

Utilization of L-rhamnose, L—mannose, and a

mixture of these L-hexoses by actively
growing L-mannose-positive cells . . .

Growth of wild-type and L—mannose-positive
cells in nutrient broth supplemented with
L-mannose e e e o e e e e o e 0

Effect of chloramphenicol and puromycin on
enzyme induction and hexose utilization
in the mutant after four generations of
growth in the absence of inducer . . .

ix

Page

115

119

125
128
130
133

136

138

140

143

145

147

150

156

Figure Page

48. Effect of chloramphenicol and puromycin
on enzyme induction and hexose utiliza-
tion in the mutant after seven generations
of growth in the absence of inducer . . . . 158

49. Growth of A, aerogenes on glycerol . . . . . 162
50. Growth of A, aerogenes in nutrient broth
supplemented with varying amounts of DL-

glyceraldehyde . . . . . . . .. . . . 165

51. Growth of A, aerogeneg on L-galactose. . . . 168

INTRODUCTION

Aerobacte£_aerogenes PRL-R3 readily gains the ability
to utilize rare hexoses, pentoses, and pentitols such as
L—lyxose (l), L—xylose (1,2), Learabitol (3), xylitol (3),
D-lyxose (4), and D-allose (5) as sole sources of carbon
and energy. Investigations notably in the laboratories of
Lin (6),‘Wood (7), and Mortlock (8,9) have shown that the
biodegradation of these rare compounds does not involve
the synthesis of new enzymes, but rather the selection of
constitutive mutants containing high levels of non-specific
enzymes capable of converting the uncommon sugars into
readily metabolizable intermediates.

The purpose of this present investigation was to
elucidate the biodegradative pathway of the unnatural hex-
ose Lemannose in A, aerogenes PRL-R3, to characterize the
enzymes involved in the pathway, and to establish the basis

for the gain in the ability to grow on this hexose.

PART I
The Biodegradation of LeMannose

by Aerobacter Aerogenes

Aerobacter aerogenes PRL-R3 readily gains the ability
to utilize L-mannose as a sole carbon source. Except for
a recent report (10) that liver galactose dehydrogenase
oxidizes L-mannose, the metabolism of this sugar has not
been previously described for any organism. This portion
of the thesis elucidates the biodegradative pathway of L-
mannose in A, aerogenes PRL-R3.

EXPERIMENTAL PROCEDURE

growth of Cells and Preparation of Extracts- A strain
of A, aerogenes PRL-R3 selected for its ability to grow
readily on L-mannose was used. The selection of this
mutant will be described in Part III. It was grown aero-
bically at 30° in a medium consisting of 0.71% Na2HPO4,
0.15% KH2P04, 0.3% (NH.)250., 0.01% M9804, 0.0005% FeSO.‘
7H20, and 0.4% sugar (autoclaved separately). The sugar
was L-mannose unless noted otherwise. The cells were
harvested by centrifugation after 15-18 hours growth in
Fernbach flasks on a rotary shaker. They were washed with
water, suSpended in 0.02 M tris-HCl buffer (pH 7.6), and
disrupted by a 4-minute eXposure in a Raytheon 10-kc
sonic oscillator equipped with an icedwater cooling jacket.

2

The cellular debris was removed by centrifugation at
31,000 x g for 10 minutes. The resulting supernatant
was the crude extract.

Analytigalggrocedures- Reducing sugars were deter-
mined by the method of Folin and Malmrose (ll). L—Fructose
and L—fructose-l-P were determined by the method of Roe
(12); the molar extinction of L-fructose-l-P was 80% that
of fructose. Inorganic orth0phosphate was determined
by the method of Fiske and SubbaRow (l3), and total phos-
phate by the method of Umbreit, Burris, and Stauffer (l4).
Trioses were determined by the method of Sibley and Lehn-
inger (15). Reducing sugars were chromatographed on‘What-
man No. 1 paper and developed with the following solvent
systems: 1) water-saturated phenol; and 2) 2-butanone:
acetic acid:water (75:25:10). The sugars were located with
a bath of silver nitrate (l6) and Sprays of orcinol (l7)
and N,N—dimethyl-p-phenylenediamine monohydrochloride (18).
Sugar phosphates were chromatographed on Whatman No. 1
paper (washed with 2 N HCl and water) using Arbutyl alcdhol:
water:picric acid (80:20:2) as the develOping solvent (19).
The location of the compounds also was determined with a
bath of silver nitrate. Glycerol and glyceric acid were
chromatographed on Whatman No. 1 paper using ethyl acetate:
pyridine:water (l20:50:40) as the developing solvent. The

spots were located by baths of benzidine hydrochloride and

of periodate (20). Protein.was measured by the method
of Tombs, Souter, and Maclagan (21). Trimethylsilyl
derivatives of D- and L-fructose-l-P were prepared and
subjected to gas chromatography by the method of Wells
g§_§A;(22). Gas chromatography was performed on a Hewlett
and Packard gas chromatograph, model 402. Melting points
were determined with a Kofler miro-melting point appara—
tus. Optical rotations were made with a Zeiss photo-
electric polarimeter or a Bendix digital pelarimeter.
Light measurements were measured on a Coleman Junior
Spectr0photometer (18-mm diameter round cuvettes) or a
Gilford absorbance recording SpectrOphotometer (1.0-cm
light path) thermostated at 25°. Manometric measurements
were made with a conventiona1.Warburg reapirometer.
§ngymejAssay§r The reaction mixture (0.58 ml) for
Lemannose isomerase contained 16 umoles of L-mannose,
2 umoles of COClg, 8 umoles of tris-HCl buffer (pH 7.6),
and enzyme. The mixture was incubated at 30°, and samples
were removed at time intervals and assayed for L-fructose:
the values were corrected for slight interference from
L-mannose. The amounts of enzyme assayed for the time
periods of incubation were selected to give linear measure-
ments. .A unit of L-mannose isomerase was defined as the

amount that formed 1 umole of L-fructose per minute in

this assay.

The L-fructose kinase activity was measured Spectro-
photometrically at 340 nm and 25°. The reaction mixture
(0.15 ml) consisted of 1.5 umoles of L-fructose, 0.5 umole
of ATP, 1.0 pmole of MgCle, 0.5 umole of phOSphoenOl-
pyruvate, 0.01 umole of NADH, 2.0 umoles of tris-HCl buffer
(pH 7.6), 13 ug of lactate dehydrogenase-pyruvate kinase,
and a limiting amount of L-fructose kinase preparation.

A control to correct for NADH oxidase and ATTase contained
all of the reaction components except L-fructose. The
absence of erructose reductase activity in the extracts

and fractions made a control without ATP unnecessary. The
assay was linear with time and enzyme concentration. A

unit of L-fructose kinase activity was defined as the amount
that resulted in the oxidation of l umole of NADH per

minute in this assay.

LPFructose-l—P aldolase activity also was measured
spectrOphotometrically at 340 nm and 25°. The reaction
mixture (0.15 ml) consisted of 1.5 umole of L-fructose-
l-P, 0.01 umole of NADH, 2.0 umoles of tris-HCl buffer
(pH 7.6), 1 pg ofcx-glycerol phosphate dehydrogenase, and
a limiting amount of L-fructose-l-P aldolase preparation.)
A control to correct for NADH oxidase contained all of the
reaction components except L-fructose-l-P. The assay was

linear with time and enzyme concentration. A unit of L-

fructose-l-P aldolase was defined as the amount that
resulted in the oxidation of l umole of NADH per minute
in this assay.

L-Glyceraldehyde reductase activity was measured
Spectrophotometrically at 340 nm and 25°. The reaction
mixture (0.15 ml) consisted of 1.5 nmoles of L-glyceral-
dehyde, 0.01pnmfle of NADH, 2 umoles of tris-HCl buffer
(pH 7.6), and a limiting amount of reductase (45-60%
(NH4)2504 fraction).

Reagents- LmMannose'was prepared by the method of
Sowden and Fischer (23) and L-glyceraldehyde by the
method of Perlin and Brice (24). Details for the prepara—
tion of these compounds are given below. D—Fructose-l—P,
D-fructose-6-P, D-fructose-l,6-diP, and o-glycerol phos-
phate dehydrogenase were purchased from Calbiochem, Los
Angeles, California; ATP, NAD, NADH, NADP, NADPH, and
phosphoenolpyruvate from P-L Biochemicals, Milwaukee,
Wisconsin; lactate dehydrogenase-pyruvate kinase from
Worthington Biochemical Corporation, Freehold, New Jersey:
wheat genm acid phosphatase and protamine sulfate from
Sigma Chemical Co., St. Louis, Missouri. L-Fructose
for use as seed crystals was a generous gift from Profes—.
sor M.L.‘Wolfrom, Ohio State University. Duolite resins

C-25 (Na*) and A-6(ClI)‘were purchased from the Diamond

Alkali 00., Redwood City, California. They were converted
to the H+ and OH- forms reSpectively and mixed in a ratio
of 3:5 (wet weight) cation:anion prior to preparation 0f
the column.

Aggparatign of L-Mannose- LeArabinose (150 g),
300 m1 of absolute methanol, and 540 ml of mitromethane
were stirred rapidly in a 3-liter, 3-necked flask fitted
'with a mechanical stirrer and drying tube. To this was
added a solution of sodium methoxide, prepared by dissol-
ving 32 g of sodium metal in 1050 ml of absolute methanol.
The mixture was stirred approximately 20 hours during which
time the reaction mixture changed from a white to yellow-
brown suspension. The sodium Eggfnitroalcohols were col-
lected by suction filtration and washed with 450 ml each
of cold absolute methanol and cold petroleum ether B.
The nitroalcohols were dissolved in 1 liter of water at
0° and immediately were added dropwise to a stirred solu-
tion of 210 ml of sulfuric acid in 250 m1 of water at room
temperature. During the course of adding the nitroalcohol
solution, the temperature of the acid solution did not
rise above 43°. The resulting solution was diluted to
3000 ml with water and solid sodium carbonate added batch—
wise until the solution became neutral to congo red. The

mixture then was treated with a solution containing 105 ml

of phenylhydrazine in 240 ml of glacial acetic acid and
the resulting solution left for approximately 12 hours
at 4°. The Lemannose phenylhydrazone which precipitated
was collected by filtration, washed thoroughly with water,
with 95% ethanol, and finally with anhydrous ether. The
crude hydrazone amounted to 110 g, melting point 184-1860.
The L-mannose phenylhydrazone was suspended in a
solution containing 1100 ml of water, 220 m1 of ethanol,
140 ml of benzaldehyde, and 14 g of benzoic acid. The
reaction mixture was refluxed for 2.5 hours, cooled to
room temperature, and the aqueous phase decanted from the
benzyl phenylhydrazone. The solution next was extracted
three times with chloroform (500 ml of chloroform per 500
m1 of solution), decolorized with Darco G-60, and concen-
trated in a vacuum to a syrup. To crystallize the L-
mannose, the syrup was dissolved in a volume of warm
absolute methanol equal to twice the volume of the syrup.
Next, a volume of absolute methanol and iSOprOpyl alcohol
(50:50) equal to the previous volume of methanol was added.
The solution was seeded with L-mannose and left overnight
at room.temperature. The L-mannose crystallized readily,
but scratching the inside of the container with a glass

rod hastened further crystallization. The Irmannose was

collected by suction filtration and washed with a 75:25
(v/v) solution of absolute methanol and iSOprOpyl alcohol.
The filtrate was reprocessed to Obtain additional Lemannose.
The final yield of L-mannose was 45-50 9 (30-33%). The
melting point was l24-l28° and the Specific rotation was
Ealg$8 -l4.2° (g_l, water). A summary of the reactions
for the synthesis of Lemannose is outlined in Figure l.
grgparation of L-Glyceraldehyde- L—Sorbose (5.0 g)
was dissolved in 10 ml of water and the resulting solution
diluted to 500 ml with glacial acetic acid. After the
solution was cooled with an external water-bath to.l7°,
28 g of lead tetraacetate was added over a period of 5—10
minutes with good stirring. During this time the tempera-
ture did not exceed 22°. Next, a solution of oxalic acid
(5.0 g of anhydrous oxalic acid in 50 m1 of glacial acetic
acid) was added until a negative starch-iodide test was
Obtained. The insoluble lead oxalate was removed by fil-
tering the solution through filter aid. The filtrate then
was concentrated in a vacuum to a syrup. Small amounts of
acetic acid subsequently were removed by adding 20 ml of
toluene followed by concentration to a syrup in a vacuum:
this Operation was repeated several times. The syrup was'

dissolved in 25 ml of 0.1 N sulfuric acid and the resulting

solution stored for 24 hours at 35°. Finally the solution

Figure l.

SYNTHESIS OF L-MANNOSE FROM L-ARABINOSE

H- =0

H- -OH CH3N02

HO- -H -—————-

HO-g-H NaOCH3
H20H

LnArablnose

 

_———a-

 

H N02

H06 -
H...
HO-

HO-

-OH
-H
-H
H20H

1-De0xy-1-n1tr0-L-glucitol

0H N02
H-q- H
H- -OH
HO- -H
Ho-q-H
CHZOH

1) 112304
.__.2) Na2C03-——e

3) Phenyl-
hydrazine

 

1-Deoxy-1-n1tr0-L-mann1tol

-OH

-H

-H
03203

HO-

H...
HO-
HO-

L-Glucose phenylhydrazone

(soluble)

0HeNNHC6H5
H-C-OH
H- -OH
30- -H

Ho-q-H

CHZOH

L-Mannose phenylhydrazone

(insoluble)

lO

CH=0
H-¢-0H
1) C6HSCH=O H-q-OH
___> Ho-q-H
0003 Ho-q-H

2) 06H
CHZOH

5
3) 03303203

L-Mannose

11

was passed through a column of Duolite C—25(H*) and A-6(OH_)
and the neutral effluent concentrated in a vacuum to a syrup.
The syrup was dissolved in water and chromatographed on
paper using Arbutanolzethanol:water (52:32:16) as the
develOping solvent (25). The sugar was identified with
silver nitrate and showed traces of L-sorbose. Since L-
sorbose did not interfere in the enzymatic assays the
crude L-glyceraldehyde was used as such.
RESULTS

Since a variety of hexoses metabolized by A, aerogenes
PRL-R3 initially are phosphorylated with ATP, my initial
investigations on the metabolism of Lemannose‘were concen-
trated on determining whether or not Lemannose also was
phosphorylated with ATP. Preliminary investigations.
involving manometric, titrametric, and colorimetric tech-
niques supported this contention. These studies are
summarized below.

Apparent Phosphorylation of L-Mannose:
.ManometrAgiqLeMannose-Stimuigted COa_Evolution ggom
Agcagbonate in the Presence of ATP- 'Wild-type and Lemannose-
positive cells were grown on D—glucose and.1wmannose reSpec-

tively and the cell-free extracts assayed for their
ability to stimulate CO; release from bicarbonate due to

the phosphorylation of hexoses with.ATP. Since the enzymes

12

of D-glucose metabolism are constitutive, C02 rapidly
evolved when extracts from both wild type and mutant were
incubated with D-glucose and ATP (Figure 2). On the other
hand, Lemannose was metabolized rapidly only by the extract
of the mutant cells grown on Lemannose, suggesting that
L-mannose degradation involves a phOSphorylation at some
point in the pathway.

Titrametric: Increased Rate of Acid groduction from
Aggin the Presence of LeMannose- The proton released '
from the utilization of.ATT with L—mannose was titrated.
Figure 3 shows a slightly greater increase in the activity
with Lemannose compared to the endogenous ATPase. To test
the validity of the phosphorylating system, D-glucose
was added as a control.

Colorimetgic: ATgeStimulated Disgppearance offAr
Mannose- The disappearance of Lemannose in extracts of
Lemannose-grown cells was stimulated by ATP, suggesting
the formation of phOSphorylated intermediates (Figure 4).
Again, D-glucose was employed as a positive control.

SpectrOphotometringest for ArMannokinase— Attempts
to demonstrate the direct phOSphorylation of Lemannose
yielded negative results, but Figure 5 shows that incubating
L—mannose with crude extract prior to adding ATP resulted

in measurable kinase activity. This suggested that L-

13

Fig. 2. LeMannose-stimulated C02 evolution from bicar-
bonate in the presence of ATP. Each Warburg veSsel con-
tained in a volume of 0.5 ml: 10 umoles of hexose (side-
arm), 5 umoles of ATP, 25 umoles of M9C12, 5 umoles of
NaF, ll umoles of NaHC03, and crude extract (3-5 mg of
protein). The reaction was carried out in an atmOSphere
of 95% nitrogen: 5% carbon dioxide at a temperature of
30°. Hexose was omitted to measure the endogenous ATPase

activity. Controls minus ATP gave no C02 release.

Figure 2.

 

_ A I
I
neocomoecM\Iv
I
I III. 0
. mo
mmoccoZqu 1
1[/300usth
o . lo;

mHHoo cachetomonnmzlq

 

mMBDZHS

m.“ 0.". m

meocmwoocM\\llw

aromoscmztac

mHHoo sackclmmoosaolm

     
     

1!:Omoosdoln

lm.o

lot"

 

NIEIOHd Sm/zoo setomn

14

15

Fig. 3. Titrametric measurement of acid production
from.ATP in the presence of Lemannose. The reaction
mixture (1.5 ml) contained 20 umoles of hexose, 25
umoles of ATP, 50 umoles of M9C12, and crude extract
(10-15 mg of protein). The reaction was carried out
at room temperature with a Beckman Zeromatic pH meter.
The reaction rate was recorded by adding a measured
volume of 0.1 N NaOH to the reaction at a specific
time to maintain the pH at 7.5. Endogenous ATPase
activity was determined by leaving out the hexose

from the reaction. Controls minus ATP gave no rate.

Figure 3.

umoles NaOH

20

15

10

 

    
    

-O- Endogenous

-—O— L-Mannose
D-Glucose

added
\

MINUTES

16

17

Fig. 4. ATP-stimulated disappearance of Lemannose.

The reaction mixture (2.0 ml) consisted of 16 umoles

of hexose, 20 umoles of ATP, 100 umoles of MgClg, 100
umoles of NaF, 100 umoles of glycylglycine buffer, pH
7.5, and crude extract (20-25 mg of protein). The
reaction was incubated at 30° and 0.3 ml aliquots removed
at time intervals and added immediately to 0.3 m1

of 5% ZnSO4 followed by the addition of 0.3 ml of

0.3 N Ba(OH)2. The insoluble BaSO4 was removed by
centrifugation and 50 ul of the supernatant assayed

for remaining reducing sugar.

Figure 4.

umoles HEXOSE

20

15

10

 

   
  

 

.A
7‘

N\\\_L-Mannose or

D-Glucose
(N0 ATP)

é,,_..—------—L--Mannose + ATP

8,,a-D-G1uoose + ATP

20 4O 60

MINUTES

18

19

Fig. 5. Lactate-dehydrogenase-pyruvate kinase-linked
assay for the apparent phosphorylation of L-mannose
with.ATP. (Refer to Assay section on L-fructose
kinase for details). Reaction mixtures (70 pl)
containing 2.5 pmoles of Lemannose, l pmole of tris-
HCl buffer (pH 7.6), l pmole of MgCla, and crude
extract (0.024 mg of protein), were incubated for 1
hour at room temperature, after which.ATP, lactate
dehydrogenase and NADH were added and activity re-
corded (B). The left portion of the graph (A) depicts
a control in which.ATP and NADH were added at zero

time.

Figure 5.

mmBDZHS

 

 

ma OH m 0 ma 0H m
A _ _ a .1 _
.I1 m
C O .
msocowoecm.JL msocmwoecmllldw
ll 0H

omocsmth

Alll.mmoccszuq

 

 

0H

ma

GEZIGIXO HGVN sanmu

2O

21

mannose was converted to another intermediate prior to
phosphorylation. Such a conversion might conceivably
involve a phosphotransferase reaction as demonstrated
by Kamel and Anderson (26). They reported that when D-
mannose was incubated with crude extracts, D-glucose
accumulated due to phosphorylation of D-mannose with
endogenous D-glucose-6-P. In the present investigation,
however, Lemannose was found to be isomerized to L-fruc—
tose.

Isomerization of L-Mannose' to L-Fructose- L—Mannose
was incubated with crude extracts and the mixture chro-
matographed on paper. Figure 6 shows the accumulation of
a spot which co-dhromatographed with authentic D-fructose
and gave a positive orcinol test suggesting that L-
mannose undergoes an isomerization to L-fructose. The
preparation and isolation of the product of Lemannose
isomerization is described below.

Enzypic Preparation of L—Fructose- The reaction
mixture (75 m1) consisted of the following: 28 mmoles of
L-mannose, 5 mmoles of COC12, l2 mmoles of tris-HCl buffer
(pH 7.6), and cell—free extract (300-350 mg of protein).
The reaction was incubated at 300 for 2-3 hours or until
equilibrium was established. At equilibrium, the ratio

of LemannosezL-fructose was 38:62 (Figure 7). The reaction

22

Fig. 6. Chromatography of the products of Lemannose
isomerization. The reaction mixture (1.4 ml) con-
sisted of 20 pmoles of Irmannose, 100 pmoles of
-glycylglycine buffer (pH 7.5), and a volume of crude
extract containing 20 mg of protein. The reaction
was incubated at 30° and 0.6 ml aliquots were removed
at time intervals (t), heated in a boiling water-bath,
and the denatured protein removed by centrifugation.
The supernatant was concentrated in a vacuum to about
50 pl and a volume of this solution containing about
0.5 pmole of hexose was Spotted on paper and then

deve10ped in.water-saturated phenol for 24 hours.

Figure 6 .

ooHHOHSoosomsosoa ocdsedo
locoHASosaIQIHASpoaaouz.z

\<</\<<<<</>7

 

o

=
t

oASpHHE
codpoeom

t=60 min.

 

 

D-Fructose
D-Glucose
L-Mannose

 

 

AOZHomo

\\(\()\)§(\/\/\/>S</)

mos:

 

 

t=60 min.
0

=
t

chopxaa
Soapowom

 

D-Fructose

D-Glucose

L-Mannose

 

 

 

A o
=

t
mASpHas

capoemm

t=60 min.

D-Glucose

L-Mannose

 

23

24

Fig. 7. Equilibrium of the Lemannose Z! L-fructose
interconversion. The reaction mixture (1.16 ml) con-
sisted of 10 pmoles of Lemannose or L-fructose, 6
pmoles of COClg, 20 pmoles of tris-HCl buffer (pH 7.6),
and crude extract (4.4 mg of protein). The mixture was
incubated at 30°, and at time intervals samples were

removed and assayed for fructose.

Figure 7.

mmBDzHE
omH oma om o:

 

 

_ . 1 a _

 

 

mmOBODmmIA AEHF mmOZZQSIA

SDHmmHAHDGM

_ _ _ _

    

 

 

om

Goa

ESOIOHHJ'T %

25

26

mixture then was heated in a boiling water-bath for
several minutes, and the denatured protein removed

by centrifugation at 32,000 x g for 15 minutes. Next,
Irmannose‘was separated from L-fructose by adding a
solution containing 3.1 g of phenylhydrazine in 7.0 m1
of glacial acetic acid and leaving the resulting mixture
overnight at 4°. The Lemannose phenylhydrazone which
crystallized was removed by suction filtration. The
filtrate then was reduced under vacuum to about 50-60 m1
and stored an additional 2-3 hours at 4°. The precipitate
that formed was removed by suction filtration. Free
L-fructose was regenerated from the phenylhydrazone by
refluxing the filtrate for 2-3 hours with 13 ml of ethanol,
8 ml of benzaldehyde: and 0.8 g of benzoic acid. The
liquid phasee,after being cooled to room temperature,
was decanted from.the insoluble benzyl phenylhydrazone,
washed with three lOO-ml portions of chloroform, and
decolorized with Darco G-60. This solution (about 60 ml)
was deionized by passage through a column of mixed-

bed resins consisting of Duolite C-25 (H1) and A-6 (0H3)
(2.7 cm x 30 cm). About 500 ml were collected, which
yielded a 96% recovery of L-fructose. The neutral
effluent was reduced in a vacuum to a syrup. L-fructose

was crystallized as fine needles by dissolving the syrup

27

in warm absolute alcohol, seeding with crystals of L-
fructose, and leaving at room temperature for 24 hours.
The crystals were collected by gravity filtration and
the mother liquor reprocessed to give additional product.
The overall yield of L—fructose was 1.4-1.6 g (28-32%),
m.p. 93-950, [uglier 91.00 (g 2.5, water) (27). Figure
8 is a photograph of the L-fructose crystals and Figure
9 is an outline of the procedure for the preparation of
L-fructose.

grepapation of L-Glucose Phenylosazone- Both fructose
and glucose produce the same osazones when heated with
excess phenylhydrazine. The osazone derivative was
prepared by the procedure of Gerard and Sherman (28).
The reaction mixture (10.0 ml) consisted of 0.1 g of
L-fructose, l g of phenylhydrazine, 1.25 g of glacial
acetic acid, and 0.68 g of sodium acetate. The pH of
the resulting mixture was 4.0-4.2. The reaction.mixture
was heated in a boiling water-bath for 30 minutes during
which time bright yellow needles of L-glucose phenylosa-
zone appeared. The product was collected by gravity
filtration, washed with five lOeml portions of water,
and dried in an oven at 100°. This same procedure also
'was used to make D-glucose phenylosazone from D-fructose

and D-glucose. All of the products gave a melting point

28

Fig. 8. Crystals of L-fructose. The crystals were
obtained from the first cr0p crystallized from abso-
lute alc0hol. They were photographed under an A0
Spencer phasestar microsc0pe fitted with an ortho-
illuminator. They were photographed with Kodak

35 mm.Panatomic-x film through the 43x objective,
giving a magnification of 860x on the film. Final

magnification on the photograph is 3,600x.

Figure 8.

 

29

Figure 9.

ENZYMATIC PREPARATION OF L-FBUCTOSE
GENERAL OUTLINE

L-Mannose

I CC“
Isomerase

i

L-Fructose

 

 

 

1) heat
2) deionize
v
Denatured protein L-Mahnose
(insoluble) and
L-Fructose
(soluble)
phenylhydrazine
(pH 4)
l
L-Mannose L-Fructose
phenylhydrazone phenylhydrazone
(insoluble) (soluble)
Benzaldehyde
(reflux)
v

 

L-Fructose

30

A. 31

of 205-208° in good agreement with the reported values.
Appparation of Phenyl—L-glucosotriazole- The
osazones previously prepared were converted to the tria-
zole derivative by the method of Hann and Hudson (29).
The reaction mixture consisted of 0.1 g of L-glucose
phenylosazone, 0.3 g of cepper sulfate, 0.5 cc of 0.5
N H2804, 6 ml of methyl alcohol, and 9 ml of water.
The mixture was refluxed for 2 hours during which time
' the color of the reaction changed from a deep red-brown to
yellow-green. The solution was cooled and then concen-
trated on a steam-bath to about 4-5 ml. After cooling
in the refrigerator for 3 hours a brown precipitate
formed which.was collected by gravity filtration, washed
thoroughly with water, and then dissolved in 15 m1 of hot
water. The solution was decolorized by Darco G-60,
filtered hot, and left at 4° overnight. The white needles
which crystallized were collected by gravity filtration
and dried in an oven at 90°. The phenyl—L-glucosotriazole
had a melting point of 193-1970 and a [a]§$8,= +80.0°
in good agreement with reported values.
‘Wa;p£_Content of L-Fructose- The enzymatically
prepared L-fructose was essentially anhydrous as was
also reported by‘Wolfromf(30).onable I summarizes the
per cent loss in weight of water from L-fructose, and

commercial D-fructose and L-rhamnose (monohydrate) in

32

TABLE I
Water content of L-fructose

The water content of L-fructose was determined in an Abderhalden
drying apparatus. D-Fructose (Pfanstiehl) and L-rhamnose (General
Biochemicals) were used as standards. The solvents, £:butyl alcohol
(b.p. 81°) and toluene (b.p. 110°), were Mallinckrodt reagent grade
chemicals. The contents of the tube were heated at solvent tempera-
tures for two hours. The samples then were cooled to room temperature
and weighed on.a Mettler analytical balance. None of the samples

possessed hygroscopic properties.

 

 

 

Solvent C und Initial Weight Final Weight Loss in % Loss
ompo (mg) (mg) Weight (mg) in Weight
.g-butyl L-fructose 98.9 97.6 1.3 1.3
alcohol
D-fructose 197.9 197.9 0 0
L-rhamnose
(monohydrate) 200.2 190.8 9.4 4.7
toluene L-fructose ‘ 81.0 79.8 1.2 1.5
L-rhamnose

(monohydrate) 199.2 187.6 11.6 5.7

 

33;

the presence of two different boiling-point solvents.

The water content of fructose influences its crystal-
line form. D-Fructose crystallizes as fine needles of
the hemihydrate which, when vigorously shaken at 0°,
is converted to prisms of D-fructose dihydrate (31).
Seeding the hemihydrate above 20° with anhydrous D-
fructose changes the needles to prisms of the anhydrous
form (32). Although anhydrous D-fructose does not
appear to crystallize as needles, anhydrous L—fructose
does (30).

ggowth of A. aegggenes ongp;§ructose- The Lemannose-
positive strain, unlike the wild-type, is capable of
growing on L-fructose as the sole carbon source (Figure 10).

ghogphorylation of L—Fructose to L-Fructose l-
PhOSphate- The L-fructose enzymatically prepared from
Lemannose was used to determine the next reaction in the
pathway. Since the utilization of ATP in crude extracts
still had not been explained, investigations were conducted
to determine if L-fructose was the phOSphorylated interme-
diate. The results showed that crude extracts from L-
mannoseegrown cells possessed a kinase which phosphorylated
L-fructose with ATP. The isolation and characterization

of the phosphorylated product is described below.

34

Fig. 10. Growth of A, aerogenes PRL-R3 on L-fructose.
A suSpenSion (0.05 ml) of either wild-type or L-
mannose-positive cells previously grown in nutrient
broth Were inoculated into 7.0 ml of mineral medium
supplemented with L-fructose (0.28%). The cells

were allowed to grow at 30° on a reciprocating shaker
and growth was recorded by measuring the increase

in the turbidity of the culture at 540 nm with a

Coleman Junior Spectr0photometer.

Figure 10.

OoDesuo

0.8

0.6

0.4

0.2

 

  
 

I l l
l-—
L..—
L-Mannose
positive mutant‘\'s
k(,—Wild Type

   

 

 

 

O 20 4O 60

HOURS

35

36

‘Enzypic Preparation of L-Fructose l-PhOSphate- L-
Fructose l-phOSphate was prepared by the following pro-

 

cedure: the reaction mixture (100 ml) consisted of 1.0
mmole of Lpfructose, 1.2 mmoles of ATP, 1.2 mmoles of
MgCla, and 7-fold-purified L-fructokinase (8.2 mg of
protein): details of the purification procedure are
outlined in Part II of this thesis. The reaction was
carried out at 22° and at a pH of 7.5. The extent of
phosphorylation was determined by automatic titration
(Sargent recording pH-stat) with 0.5 N NaOH. After 8
hours the reaction was complete. The resulting mixture
‘was chilled in ice, 2.4 mmoles of barium acetate were
added, and the resulting mixture was left an additional
30 minutes in the ice bath. The precipitate that formed
was removed by centrifugation. The pH of the supernatant
‘was then brought to pH 2.0 with hydrobromic acid and the
260 nm—absorbing material removed by treatment with
Darco G-60. The pH was increased to 8.0 with NaOH and
the solution cooled in an ice bath. Cold ethanol then
was added to a final concentration of 80% and the resul-
ting solution left at ”0 overnight, during which time a
‘white flocculent precipitate of barium L-fructose 1-
phosphate fonmed. The precipitate was washed with 80%,

90%, 95%, and absolute alcohol and then with anhydrous

37

ether. The final yield was 322 mg (81%). Figure 11
outlines the procedure.

Identification of_A:Fructose l-phosphape:

(a) Pappr Chromatography- The barium salt of the
product was deionized by Dowex 50W-X8(H1) and the free
sugar-phosphate chromatographed on paper. The product
co-chromatographed with D-fructose l-phOSphate and was
distinguishable from both D-fructose 6-phOSphate and
D-fructose 1,6-diphosphate.

(b) gructose to phosphorus Rapipr The fructose to
phOSphorus ratio was 1 to l and the amount of phosphate
released after 80 minutes hydrolysis in l N H2804 was
equivalent to that released when the sugar—phOSphate
was hydrolyzed by 10 N H2804 (Table II).

(c) Polarimetry- The product had a Specific rotation
of-+50.7° compared to -5l.0° for D-fructose l-phOSphate.

(d)‘flheat Germ.Acid Phosphatase- 'When the product
was dephOSphorylated by wheat germ acid phOSphatase and
Chromatographed on paper, a single Spot appeared which
co—chromatographed with authentic D-fructose in two solvent
systems.

(e) Acid Labile PhOSphate- Figure 12 compares the
rate of phOSphate released when the product was hydrolyzed

by l N H2804. Both L- and D-fructose l-phosphate are

Figure 11.

ENZYMATIC PREPARATION OF L-FRUCTOSE-l-PHOSPHATE
GENERAL OUTLINE

L-Fruotose

ATP
Mg++

kinase

 

L-Fruotose-l-Pop

 

 

 

Barium
acetate
Insoluble Barium Soluble L-Fruotose-iqPOuBa
salts nucleotides
1) pH 2
2) Darco G-6O
nucleotides L-Fruotose-l-Pou
(Ba salt)
pH 8
' EtOH to 80%
Insoluble

L-Fruotose-l-POuBa

37'a

38

TABLE II
Phosphate to fructose ratio of L-fructose-l-P
Aliquots of L-fructose 1-phosphate were added to 4.0 m1 of 1 N H2304
and the solution placed in a boiling water-bath for 80 minutes. The
solution was cooled and assayed for total free fructose and phosphorus.
Fructose was determined by the Roe test. Inorganic phosphate was

measured by a modified procedure of Fiske and SubbaRow.

 

*7

 

Compound assayed Amount

 

singles

‘Aefore dephosphorylation:
L-Fructose-l-P 0.88
Total Phosphate 0.86
After dephosphorylation:
L-Fructose 0.86

Inorganic Orthophosphate 0.85

 

39

Fig. 12. Acid hydrolysis of D-fructose-l-P, D-
fructose-6-P, and the product of Lpfructose phos-
phorylation. Tubes containing 1.0 pmole of the
indicated fructose ester in 4.0 ml of l N H2804.
were heated in a boiling water-bath. At time
intervals, they were removed and the contents

assayed for inorganic orthophosphate.

Figure 12 .

 

 

 

 
  
  

1 F l
100 — 3
-o,- D-Fruc toss-1 -P
-.-L-Fructose-1-P
75 e
to
H
3’.
r-‘l
o
m
E 50 ..
t9.
25
D-Fruotose-6-P

  

 

 

 

MINUTES

40

41

hydrolyzed at the same rate. D-Fructose 6-phOSphate
hydrolyzes at a slower rate, and on this basis the C-1
phosPhate is distinguishable from the C-6 phOSphate. The
half-life (50% hydrolysis) of both the product and D-
fructose l-phosphate was 8 minutes.

(f) ggg Chromatogrgphy- The gas chromatography
patterns of the trimethysilyl derivatives of D- and L-
fructose-l-P are similar (Figure 13).

Enzypic Cleavage of LPFructose leghogphate- The
metabolism of L-fructose 1-ph08phate undergoes a direct
cleavage to dihydroxyacetone phosphate and L-glyceralde-
hyde. Figure 14 Shows a direct relation between the rate
of cleavage of L—fructose l-phOSphate and the rate of
formation of trioses. Dihydroxyacetone phosphate was
identified by coupling to NADH linked cr-glycerol phos—
phate dehydrogenase. The rate at which dihydroxyacetone
phOSphate forms also is an indication of the aldolase
activity (see assay section on aldolase). The other
cleavage product has not been Specifically identified
but would be exPected to be L—glyceraldehyde. Cleavage
of L-fructose-l-P by either crude extracts or the
partially purified aldolase fraction could not be detected
in the absence of a coupling system to remove one of the

products, indicating that the equilibrium of the reaction

42

Fig. 13. Gas chromatography: preparation of the
trimethylsilyl derivatives of fructose-l-P. A solu-
tion containing 600 pg of the phosphorylated pro-
duct of L-fructose was concentrated in a vacuum and
the residue dissolved in 2.0 ml of methanol and
diluted with 2 ml of diethyl ether. The phos-
phoric acid group was esterfied with diazomethane
generated from N-methyl-N-hitroso-p-toluenesu1-
fonamide. The resulting mixture was concentrated in-
a vacuum and the residue dissolved in 10 m1 of
pyridine. Trimethysilylation was carried out by
adding 0.2 ml of hexamethyldisilazane and 0.1 ml
of trimethylchlorosilane. About 3 pg of the

product was used for a sample run (33).

Figure 13.

 

L—Fructose-l-P

 

43

 

 

 

 

D-Fructose-l-P

 

44

Fig. 14. Triose formation from L-fructose-l-P.
The reaction mixture (0.9 m1) consisted of

3.5 pmoles of L-fructose l-phosphate, 20 pmoles
of hydrazine sulfate, 80 pmoles of glycylglycine
buffer, pH 7.5, and crude extract (10-12 mg of
protein). The reaction was incubated at 30°
and at time intervals 0.1 ml aliquots were
removed and assayed for Lpfructose l-phoSphate
(14).

(O.D. (11) and trioses (O.D.

520) 540)

Figure 14 .

OPTICAL DEN SITY

0.4

0.3

0.2

0.1

 

 

 

 

l l l
__+

O
kTrioses _

O

KL-Fruotose-l-P _.

J I l

10 20 30

MINUTES

45

46

lies in the direction of synthesis. That the equili-
brium strongly favors the fructose ester also has been
noted for D-fructose-l-P aldolase (34).

Test for Possible Epimerization- The possibility
that epimerization of L-fructose-l-P to some other ketose-
l-P preceded cleavage was ruled out as follows. L-
Fructose-l-P (10 pmoles) was incubated at 25° with 1.0
ml of partially purified L-fructose—l-P aldolase (2.4 mg
of protein) in 0.02 M tris-HCl buffer (pH 7.6). After
2.5 hours, HQSOp was added to a final concentration of
l N, and the precipitated protein was removed by centri-
fugation. The supernatant solution was heated in a
boiling water-bath for 1 hour, cooled, and deionized
by passage through a mixed—bed of Duolite C-25(H+)
and A-6(OH3) (35). The neutral effluent was concentrated
;p_ypppp.and Chromatographed on paper. Visualization
of the Spots with orcinol revealed a single Spot which
co-chromatographed with fructose but not with tagatose,
sorbose, or psicose. Thus, it may be concluded that
L-fructose-l-P is the substrate for the aldolase and
is not epimerized to some other ketohexose-l-P prior to
cleavage.

A, aerogenes degrades Irmannose into products which
the cell can readily use for energy and growth. Presum-

ably dihydroxyacetone phosphate is further metabolized

47

via triose phosphate isomerase. On the other hand,
L-glyceraldehyde was degraded by at least two distinct
pathways: 1) reduction to glycerol: and 2) oxidation
to glyceric acid.

Reduction of LPGlyceraldehyde to Gcherol- An
enZyme was detected in ammonium sulfate fractions of
the crude extract whidh reduced Lpglyceraldehyde to
glycerol. A large scale incubation mixture (0.8 ml)
consisting of 20 pmoles of Leglyceraldehyde, 30 pmoles
of NADH, and about 10 mg of protein from the 40-60%
(NH4)2804 fraction was incubated at 30°. )At zero time
and at 60 minutes, incubation samples were withdrawn and
placed in a boiling water-bath. The denatured protein
‘was removed by centrifugation and the supernatant con-
centrated in a vacuum to about 50-100 pliters. Aliquots
were Spotted on paper and the chromatogram deve10ped
(See EXPERIMENTAL PROCEDURE). In the reaction mixture
incubated for 60 minutes, three Spots appeared which
co-chromatographed with glycerol, glyceraldehyde, and
glyceric acid.

Oxidation of L-Glyceraldehyde to Glyceric Acid-

The presence of glyceric acid as another product of the

 

reaction indicated the presence of another enzyme in

the ammonium sulfate fraction which oxidized Leglyceral-

48

dehyde to Lpglyceric acid. To further Show that glyceric
acid was also a product of L-glyceraldehyde metabolism
a large scale incubation mixture containing Lpglyceral-
dehyde, NAD, and enzyme was incubated as previously
described. The results showed that when NAD was present,
glycerol and glyceric acid accumulated, indicating a
dismutation existed in the metabolism of L-glyceraldehyde
in which NADH and NAD were interconverted by the enzymic
oxidation and reduction of Leglyceraldehyde.
DISCUSSION

The pathway of Lemannose metabolism in A, aerogenes
as deduced from experiments presented in Part I of this
thesis is depicted in Figure 15. L-Mannose is initially
isomerized to L-fructose, which is phosPhorylated with
ATP by a kinase to L-fructose l-phosphate. L-Fructose
l-phOSphate is then cleaved by the action of an aldolase
to yield dihydroxyacetone phOSphate and L-glyceraldehyde.

The sequence of reactions reported here is similar
to the sequences involved in the degradation of the
naturally occurring 6-deoxy hexoses, L-rhamnose and L-
fucose, in that they too undergo sequential isomerization,
phOSphorylation at carbon atom one, and cleavage (36-43).
However, no underived aldohexose other than,Lemannose

has been reported.to be metabolized through such a sequence

 

menseeaaemewaeuq

momma
_
muouom
_
om

\\

0

meme .emmaoeaa,

momma
.

ouo

_
«mnemommo

 

Figure 15.

 

mlalomOHOTHMIH

mom

MD

are.
mtmlom

melmtm

momommo

mm

 

 

on on osamwm

++we .eee

 

A.emmcam

 

omoccmznq

momma

_
mlotom
_
++oo.r mlolom
Aw _
odeoBomH monoum

_
mOIOIm
. .9
tom

 

o

mmzmwommd.mmaodmommd ZH ZOHB<m¢m0mn mmOZZdSIA mo Hdzma<m

49

50

in any organism.

The enzymic isomerization of L—fructose from Le
mannose is a convenient method for the preparation of this
rare ketohexose. L-Fructose first was synthesized
by Fischer (44) , who treated (z-acrose with phenylhydra-
zine. The resulting D,L-glucose plenylosazone was
isolated, hydrolyzed to the osone, and then reduced to
D,L~fructose. This subsequently was digested by yeast
to yield a solution of Lefructose which was identified
by its L-glucose phenylosazone, but was not, itself,
isolated in crystalline form. LPFructose was later syn-
thesized by Wolfrom and Thompson (30) by the following
sequence 'of reactiOns: L-arabonic acid tetraacetate...
L—arabonyl chloride tetraacetate_.l-diazo-l-deoxy-Apppr
L-fructose tetraacetate—+AppgrL-fructose pentaacetate-+
Lpfructose. The L-fructose was crystallized and iden-
tified by polarimetry, derivatives, and elemental analy—
sis. Tb date this procedure is the only satisfactory
method available for the synthesis of L-fructose.

The enzymic method for the preparation of erruc-
tose described here in this thesis is conSiderably
shorter than the chemical method previously described
(30). It involves only the isomerization of Lemannose,

a compound which can be readily prepared or purchased;

51

the enzyme does not need to be purified, but can be used
directly as it occurs in the cell-free extracts. Unreac-
ted Irmannose may be recovered from the equilibrium
mixture by regeneration from its phenylhydrazone. The
yield of L-fructose (28—32%) is equal to or better than
that obtained with the chemical method starting from L-
arabonic acid tetraacetate (30).

Although the isomerization of Lemannose to L-fructose
represents a new enzymic reaction, the isolation of L-
fructose is an adaptation of a standard procedure. Mannose
phenylhydrazone is well knpwn for its low solubility
in water: thus, it has been used to separate mannose
quantitatively from other hexoses. Sowden and Fischer
succeeded in separating Lemannose from.L-glucose through
their corresPonding phenylhydrazones (33), and I have
succeeded in separating Lemannose from Lpfructose by the
same means. TWO precautions might Specifically be noted.
First, a high excess of phenylhydrazine should be avoided
because L-fructose and L-fructose plenylhydrazone readily
react with the excess reagent to produce insoluble L-
glucose phenylosazone (45), thus decreasing the yield of
L-fructose. Second, mixed-bed resins possessing strong
anionic groups should not be used for deionizing solutions
of fructose because they cause partial isomerization to

glucose.

52

Since the extent of isomerization is based solely
on establishing equilibrium, the amount of L-fructose
that can be obtained is limited only by the amount of
Irmannose available; the time required to reach equili-
brium is inversely prOportional to the amount of enzyme.
Thus, larger quantities of L—fructose could be prepared
simply by increasing the initial reaction components.

L-Fructose l-phosphate is the intermediate in L-
fructose metabolism. This is the first report of this
sugar phosphate as an intermediary metabolite. On the

other hand, A, aerogenes metabolizes D-fructose differ-

 

ently. Hansen and Anderson have demonstrated a four-
component system in which D-fructose is phOSphorylated
‘with phOSphoenolpyruvate at carbon atom one (45a).

A second phOSphorylation with ATP and an induced kinase
yields D-fructose 1,6-diphOSphate (46) and this can be
metabolized by known reactions.

The metabolism of L-glyceraldehyde in bacteria has
not been previously reported: however, its metabolism by
A, aerogenes is similar to pathways determined in
mammalian tissue. Holldorf and coworkers (47) reported
an aldehyde dehydrogenase which oxidized L-glyceraldehyde.
to L-glyceric acid. Dawkins and Dickens (48) later showed

that Lrglyceric acid is oxidized further to hydroxypyru-

53

vate, a precursor to L-serine (49). Glyceric acid
accumulated in the growth media from both wild type and
L-mannose positive cells which utilized L-mannose.
‘Whether or not some of the glyceric acid was metabolized
by the cells is not known. The significance of glyceric
acid formation during the utilization of Lemannose by

A, aerogenes is considered in more detail in Part III of
this thesis.

The reduction of L-glyceraldehyde to glycerol has
been demonstrated previously inboth mammalian and bac-
terial sources. Hadorn EEHEA.(5O) Showed in the reverse
reaction that horse liver dehydrogenase oxidized glycerol
to mainly L-glyceraldehyde and only a small amount of
the D-isomer. Burton (51) reported that glycerol dehy-
drogenase from A, aerogenes reduced glyceraldehyde at
a rate 14% that of glycerol, but he did not Specify which
enantiomorph of glyceraldehyde was reduced. Lin and
Magasanik (52, 53) later reported the prOperties of glycer-
ol dehydrogenase isolated from.a capsulated strain of
A, aerogenes. In this case, however, Specificity studies
revealed that the enzyme did not reduce DLpglyceraldehyde.
My results showed that L-glyceraldehyde is reduced to
glycerol in fractionated extracts of A, aerogenes PRL-R3.

The reduction of dihydroxyacetone also has been demonstrated

54

in these same extracts, suggesting that glycerol is reoxi-
dized to dihydroxyacetone which can be phOSphorylated with
‘ATP to yield dihydroxyacetone phOSphate. The phosphoryla-
tion of glycerol could not be demonstrated in these studies.
It is of interest that.lrmannose has not yet been
found to occur naturally. Kleczkowski and Wierzchowski
(54) reported that Agcillus krzemienlewsk;_(Bacillus
circulans (55)) formed a polysaccharide consisting of
L-mannosyl residues, but Forsyth and Webley (56) later
reported that D-mannose was the correct enantiomorph.
.Although Irmannose does not occur naturally, it was
prepared initially by the Kiliani-Fischer cyandhydrin
synthesis (57, 58). However, it can be prepared more
conveniently by the method of Sowden and.Fisbher (22).
L-Fructose, also, has not yet been found to occur
naturally. Ahmed, Rizk, and:Hammouda (59) have reported
that L-fructose is a constituent of several Species of
Egyptian Plantago; however, because the sugar was iden-
tified only by chromatography on paper, a procedure
which does not distinguish between the D and L enantio-
morphs, evidence for the natural occurrence of L-fructose
must be considered nil. There also are reports that L-
fructose is phosphorylated with.ATP by cell extracts of

trypanosomes (60, 61), but this seems to be in error; a

55

close inSpection of the data reveals that D—fructose

must~have been intended.

PART II
Common Identity of the Enzymes of

LeMannose and L-Rhamnose Metabolism

Part I of this thesis showed that in.A, aerogenes
PRL-R3 Irmannose was metabolized by sequential isomeri-
zation, phOSphorylation at carbon atom one, and cleavage.
This sequence of reactions is similar to the sequence
by which Lprhamnose (6-deoxy-L—mannose) has shown to be
degraded in ggsteurella pestis (36), Lactobacillus
piantarum (37, 38), and Escherichia coli (39-41). In
each of these cases, however, the enzymes involved in
the degradation of L—rhamnose either did not degrade L-
mannose or the investigators did not use L-mannose or
its metabolic intermediates as test substrates. Part II
of this thesis describes the characteristics of the first
three enzymes of the Irmannose degradative pathway in

A, aerogenes PRL—R3 and confirms that in this organism

 

both Lemannose-and Lprhamnose are metabolized by the
same enzymes.
EXPERIMENTAL PROCEDURE
Growth of Cells and Preparation of Axtracts- Unless
otherwise stated, the cells were grown aerobically 10-12
hours on mineral medium containing 0.4% Lprhamnose. The

cell extracts were prepared as described in Part I. The

56

57

isomeraseless mutant (B-22), obtained by mutagenesis with
ethyl methanesulfonate, was provided by Dr. R.L. Anderson.

Analytical Procedures- LPRhamnulose was determined
by the method of Dische and Borenfreund (62); 0.1 pmole
of L-rhamnulose in 7.4 m1 of solution gave a O'D'540 =
0.28 (l8-mm.light path) after 10 minutes incubation at
37°. Protein was determined in crude extracts as des-
cribed in Part I and in fractionated extracts was deter-
mined by the ratio of the absorbances at 260 nm and 280
nm (63).

Enzyme Assays- The enzymatic assays were the same
as those for L—mannose metabolism, as described in Part I,
except for the following changes: for L—rhamnose isomeri-
zation the assay contained 16 pmoles of L-rhamnose and
2 pmoles of MDClg in place of L-mannose and COClg; the
kinase assay contained 0.5 pmole of Lprhamnulose in place
of L-fructose; and the aldolase assay contained 1.0 pmole
of L-rhamnulose l—phosPhate in place of L—fructose 1-
phosphate.

Partial Purification of LPFructose Kinase andggr
Fructose—l—P Aldolase- All procedures were carried out
at 0—40. The crude extract was diluted with 0.02 M tris-p
HCl buffer (pH 7.6) to give a protein concentration of

10 mg/ml. Crystalline ammonium sulfate was added to a

58

concentration of 0.1 M. A 2% solution of protamine
sulfate (pH 7.6) then was added slowly with stirring

in an amount equalling 20% (v/v) of the extract. After

10 minutes the precipitate was removed by centrifugation
and discarded, and the supernatant solution was fraction—
ated with crystalline ammonium sulfate. The protein
precipitating between 40% and 60% saturation was dissolved
in 0.02 M tris-HCl buffer (pH 7.6) and was chromatographed
on a column of Sephadex G-100 equilibrated with the same
buffer. The fractions containing the highest Specific
activity of each enzyme were combined. Both enzymes were
purified about 7-fold with a 20% recovery.

Agagents- L-Rhamnose was purchased from General
Biochemicals. Protamine sulfate was purchased from.Sigma
Chemical Co. L-Glucose was prepared by the modified
procedure of Hudson (64) and Frush and Isbell (65).
L-Rhamnulose and Lprhamnulose-l-P were prepared enzy-
matically by the following procedures:

preparation of L-Rhamnulose- L-Rhamnulose was pre-
pared enzymatically from L-rhamnose. The reaction mixture
(40 ml) consisted of 2.8 mmoles of Lprhamnose, 0.07 mmole
of MnCla, and a volume of crude extract containing about
200 mg of protein. The reaction mixture was incubated

at 45° until equilibrium was established (Figure 16),

59

Fig. 16. Equilibrium of the L-rhamnose :3 L-rhamnulose
interconversion. The reaction mixture( 0.6 ml) con-
sisted of 2 pmoles of L-rhamnose or L—rhamnulose, 3
pmoles of MHClg, 8 pmoles of tris-HCl buffer (pH 7.6),
and crude extract (1.1 mg of protein). The mixture

was incubated at 30° and at time intervals samples

were removed and assayed for rhamnulose.

Figure 16.

mmBDsz

 

 

 

 

 

0: on om 3 o

. _ _ 1
tom
L 0.:
Joe

omoaocemcmaq MIII..|/ omocaeszH 1 ow
O
ooa
SDHmmHAHDGm
_ _ _ _

 

HSOTQNNVHH’T %

60

61

after which the solution was heated in a boiling water-
bath for several minutes and the denatured protein re-
moved by centrifugation. The supernatant was decolorized
by treatment with Darco G—60 and concentrated in a vacuum
to about 3.0 ml.

The L-rhamnulose was separated from L-rhamnose by
paper chromatography as follows: 13-15 pmoles of crude
L-rhamnulose were streaked across the base line of What-
man No. 3 paper, then deve10ped 32 hours in Arbutanol:
ethanol:water (52:32:16). Strips were cut from the sides
and center of the paper and after identifying the L-
rhamnulose with silver nitrate (16), the L-rhamnulose
band was removed and washed from the paper with water.
About 90% of the L-rhamnulose, which was free from L—
rhamnose, was recovered by this procedure.

greparation of L-Rhamnulose l-Phosphate- L-Rhamnu-
lose-l-P was prepared by the method of Chiu and Feingold
(66). The reaction mixture (40 m1) consisted of 0.5
mmole of L-rhamnulose, 0.7 mmole of ATP, 0.25 mmole of
MgClz, and about 8 mg of partially purified kinase (refer
to enzyme purification for details). The reaction was
performed at room temperature on a Sargent recording
pH-Stat; the pH of the reaction was maintained at 7.5

by the addition of 0.2 N NaOH. ‘When phOSphorylation

62

was completed, L-rhamnose was separated from L—rhamnulose-
l-P by placing the reaction mixture on a Dowex—l-formate
column (24 cm x 1.5 cm). The column was deve10ped by
gradient elution with 1 liter of a solution containing
0.4 N formic acid and 0.1 N sodium formate in the reser—
voir connected to a mixing chamber containing 200 ml of
water. 50-ml fractions were collected and assayed for
both L-rhamnulose (62) and L~rhamnulose-l-P (measured
by the inorganic orthOphOSphate released (13)). Figure
17 is the elution pattern of L-rhamnulose and L-rhamnu-
lose-l-P from the column. The fractions containing the
sugar—phOSphate were pooled and concentrated to about
40 ml. The pH of the solution was adjusted to 6.4 with
saturated barium hydroxide, after which 400 ml of ethyl
alcohol was added [80% (v/v)] and the resulting solution
left overnight at 4°. The precipitate that formed was
centrifuged, washed with 80% ethyl alcohol, and anhydrous
ether, then dried in a vacuum. The final yield of
barium L-rhamnulose-l-P was 175 mg.
RESULTS

‘Whole Cell Fermentation- Cells grown on L-rhamnose
or L-mannose readily oxidized both of these hexoses at
approximately equivalent rates, whereas cells grown on

D-glucose or nutrient broth did not oxidize either L-

63

Fig. 17. Fractionation of L-rhamnulose-l-P on Dowex—
1-formate. L-Rhamnulose-l-P was purified by fraction-
ation on a column of Dowex-l-formate. It was eluted
from the column by a formate-formic acid gradient.

Details of the procedure are given in the text.

Figure 17.

 

160——

  
  

Laflhamnulose-l-P
120 \\‘~*

L-Rhamnulose

‘80 <?//

pmoles SUGAR

.-4o

 

O

l )

_—,J_

o 206 400 600

MILLILITERS

64

 

65

hexose (Figures 18 and 19).

Induction of Enzymes by L-Mannose and L-Rhamnose-
The isomerase, kinase, and aldolase activities were
determined in crude extracts obtained from cells grown
either on L-mannose or Lprhamnose (Table III). The ratio
of the rates of L-rhamnose to Lemannose isomerization
was 4:1 in crude extracts of cells grown on either L-
hexose. The ratio of the rates of L-rhamnulose to L-
fructose phOSphorylation was 1:1.1, and the ratio of the
rates of L—rhamnulose-l—P to L-fructose-l-P cleavage was
about 3:1. These enzymes were not detected in cells
grown on D-glucose or nutrient broth.

Properties of L-Mannose (L-Rhamnose)_Isomerase:

(a) Activity_in Crude Extracts- The rates of iso-
merization of L-mannose and L-rhamnose were compared as
a function of isomerase concentration in crude enzyme
extracts from both the wild-type and mutant cells (Figure
20).

(b) gartial Fractionation- The L-mannose and L-
rhamnose isomerase activities did not separate when crude
extracts were fractionated with ammonium sulfate, Sephadex
G-100 (Figure 21), and DEAE—sephadex (Figure 22). On
Sephadex G-100 the highest isomerase activity appeared

in the highest protein fraction for both Lemannose and

66

Fig. 18 Whole cell fermentation. Each Warburg vessel
contained, in a total volume of 0.55 ml, 10 pmoles of
hexose (sidearm), 180 pmoles of potassium hydroxide
(center well), and a volume of cells (2-3 mg dry weight)
suSpended in 0.2 M potassium phOSphate buffer, pH

7.0. Water was used in place of the hexose to deter-
mine the endogenous activity. The temperature was

30° and the gas phase was air.

Figure 18.

mmBDZHz

 

 

 

moocmmoocm

omoowaoum.\\\A

omoceecmnq

WHHOU CZOHUlmmOQSTZI

_ _ _

     

IIL

I

 

A

 

hV|\|-//
/ msocmwoocm

omoscdznq

omocamnmum

mmoosHonm

MHHOU QSOHOIQMOOSlem

_ _ _.

   
       

 

 

0H

ma

0N

91130 (IHDIEM 130) Bm/sxvman 20 9164110

57

68

Fig. 19. Whole cell fermentation of D-glucose, L-
mannose, and Lnrhamnose. Details are given in Figure

18.

mmBDZHS

 

 

.me 0H m e we ea m
— . _ I'Mlll-
meocmwoosm

mmoccezlq o
msocmwoocm II n . o

omocadsmrq o

0

0
I1 0H

mmoccezaqx\\\wv o

omoosfloum.\i 11»

\v .1 ma 4/
omoosawnm . mmocamnmuq
. O
. l ON 0
mHHeo czosonspOHm pcmahpsz . maamo czonoummocamnmtq

 

 

Figure 19.

69

91130 (IHDISM 130) Sm/Exvman 20 8194110

 

70

.cwouond mo wa\cHE\uo:poud

mo moHoai mm ommmmudxo mum muaomom

NO0.0 V.*

 

 

*0 *O *O *O *O *O Suoun uflmwhusz
mo.o mo.o mm.o wm.o we.o Hw.o emoeSeAp-e
No.0 eo.o No.0 mH.o mm.o mo.H emceemS-a
m-H- m-H

omouooumuq

immoascamspoq

 

omouUSHmuA

oonooamnuuA

 

omoccmatq

omocamcnaa

 

 

eeeeoeee

 

mmmcfim

 

owmpofiOmH

 

oumuumbom

nusouu

 

 

. muomhuxw QUSHU

one ca poemwsd meekuco do women was >uw>wuo<

.uxmu ecu cw pocHHuoo mouse

umooud ou wcapuooom goose ucmHuusc no «omocamsunq aomoocmfinq segues co macaw mums mHHoo any

 

mocowouom umuomoouo< ca moewnco

 

mmocaenuua one omoccmanq mo cofiuooch

HHH mAm<H

71

Fig. 20. Activity of the isomerase in crude extracts.
The routine isomerase assay was used except that the

protein concentration was varied.

Figure 20.

 

omoccmznqtllw

mmwe QQHS

szeomm we

0

    
   

 

m4 0; we

_

omoccmzuq.llJV

mmocemmmnq \\\W

BZ<BDS

     

 

 

N.o

0.0

w.o

o.H

NIN/HSOIHX setomn

73

Fig. 21. Fractionation of the isomerase on Sephadex
G-100. Details are in the text. The volume of the

fractions collected was 5 ml.

mg PROTEIN/FRACTION

Figure 21.

 

 

o -— Protein

0 — L-Mannose

O — L-Rhamnose

h ..
_ _-\ _ ._==.=.H'-_~—"

 

30 MUTAN‘I‘
20 ~—
1 O -—
o
O
30 WILD TYPE
_

10—

 

 

 

 

FRACTION NO .

74

 

 

30

20

10

30

20

10

pmoles KETOSE/MIN/FRACTION

75

Fig. 22. Fractionation of the isomerase on DEAE-
Sephadex. A volume of isomerase previously frac-
tionated on Sephadex G-100 and containing 110 mg
of protein was placed on a column of DEAE-Sephadex
(75 mm x 15 mm) which was equilibrated with 0.05 M
phosphate buffer (pH 7.0). The column was deve10ped
with a sodium chloride gradient ranging in concen-
tration from 0 to 0.6 M. The volume of the frac-
tions collected was 10 ml. For determining iso-
merase activity the reaction mixture (1.0 ml) was
incubated 1 hour at 30° and the amount of ketose

produced was determined.

mg PROTEIN / FRACT ION

Figure 22.

20

16

12

 

 

 

o—-Protein

._.L-Mannose

o— L-Rhamnose

 

 

 

FRACTION NO.

76

100

80

60

4O

20

pmole KETOSE/HR/FRACTION

77

L-rhamnose. DEAE-sephadex inactivated the isomerase
activity 90%. This high inactivation of the isomerase
coupled to the prolonged incubation periods in the assay
to measure the remaining isomerase activity resulted in

a further decrease in enzyme activity. Thus, the activity
on L-mannose and L-rhamnose as depicted in Figure 22 was
not calculated in the linear range of the assay but rather
on the amount of ketose which accumulated after a given
time interval.

(c) §pbstrate Specificitye Only L-mannose and L-

 

rhamnose were significantly isomerized; L-ribose showed
about 1-2% activity and Dmarabinose and L—fucose were
isomerized about 1% that of Lmrhamnose in wild-type
extracts (Table IV).

(d) Metal Ion Activationg Effect of Mnhl and Cok+~
Mn7+ was the only cation that stimulated L—rhamnose
isomerization (Table V). All other ions either inhibited
or did not affect isomerase activity. On the other hand,
only Co++ stimulated Lemannose isomerization (Table VI).
All other ions either inhibited or showed no effect
toward the enzyme. Coil"+ and Mn71 exerted a mutual antag-
onistic effect on the isomerization of L-mannose and

L-rhamnose. Cohfstimulated the isomerization of meannose

by 40% and Mn7+ inhibited it by 75%. On the other hand,

.78

TABLE IV

Substrate specificity of the isomerase

 

The isomerase activity was determined in crude extracts by the

standard assay.

For the other substrates (0.028 M) 5 to 10 times as

much enzyme was employed and the incubation time was extended from

10 to 30 minutes.

Except for D» and nglucose, in which the Roe test

was employed, activity on the substrates was measured with cysteine-

 

 

 

 

 

 

carbazole.
LaRhamnose-Grown Cells leMannosecGrown Cells
Substrate Comparative Rate (%) Comparative Rate (%)
L-Rhamnose 100 100
lrMannose 25 25
D-Manno s e 0 0
L~Fucose 1 0
D-Fucose 0 0
L-Glucose 0 O
D-Glucose 0 0
L~Galactose 0 0
D-Galactose 0 0
D-Ribose 1.5 2
L-Arabinose 0 0
D-Arabinose 1 0
L-Xylose 0 0
D-Xylose 0 O

 

79

TABLE‘V

Effect of various metal ions CT the isomerization of L-rhamnose

 

Refer to Table V for details.

 

 

L4Manncse~Grown Cells

Cation L‘Rhamnose«Grown Cells
Comparative Rategjfi)

ggmparative Rate (%)

 

 

 

 

None 100 100
Mn++ 155 135
Co++ 65 70
Mg++ 100 100
Fé+++ 9o 85
Ni++ 20 16
Ca”+ 55 25
Zfi++ O O
Gu++ O O
Né+ 100 100
K+ 100 100
NH4+ 100 100

 

8()

TABLE V I

Effect of various metal ions on the isomerization of L~mannose

 

The routine isomerase assay was employed in the crude enzyme extract.
. . ~3
The concentration of each cation was 394 x 10 M. The chloride salt of

each cation was used in each case.

 

 

 

 

 

 

_L;Rhamnose»Grown Cells L-Mannose-Grown Cells
Cation Comparative Rate (%) Comparative Rate4(%)
None 100 100
Mn++ 25 25
Co++ 155 IMO
++
Mg 100' 100
+++
Fe 30 55
-H-
Ni 10 15
H
Ca 12 15
H
Zn ' O ' O
+
Na 100 100
+
K 100 100
+

NH4 100 100

 

81

+ . c n a
Mn+ stimulated Lwrhamnose isomerization by 55% and
++ . . . .
Co inhibited it by 55%.
++ ++ ,

The effects of Co and Mn concentrations on L~
mannose and Lwrhamnose isomerase activities are shown
in Figure 25. The data suggest that the enzyme binds de+

++ e .
more strongly than Co' 0 However, it should be kept in
mind that crude extracts contain nucleotides and other
proteins which also may bind these metals, thus affecting
their availability to the isomerase.
++ _ ++ . . ++

When Mn and Co were mixed together, Co exerted
a greater effect on the isomerization of L-rhamnose (Table
VII). By increasing the concentration of Mn++ up to
5 times that of Co++ no significant increase in the
activity occurred. With L-mannose as the substrate,
increasing the concentration of Co+f increased L-mannose
+

. . . a , . +
isomerization and increaSing the concentration of Mn

decreased it.

 

(e) Effect of Mixing Substrates— Isomerase activity
on mixtures of Lemannose and Lorhamnose was less than
onLamannose alone (Table VIII).

(f) An Isomeraseless Mutant (B‘22)- Table IX shows
that both L-mannose and Lprhamnose induced all three
enzymes in the wildwtype cells. In the B-22 mutant only

the kinase and the aldolase were induced: the isomerase

. . + 1* ++
Fig. 25. Effect of varying the Mn and Co concen~
trations on isomerase activity. The routine isomerase
assay was used to determine activity in crude extracts.
Only the concentrations of Mn and Co were varied.
As a control the isomerase activity was measured in

the absence of the cations.

Figure 25.

umoles L-RHAMNULOSE/MIN/mg PROTEIN

umoles L-FRUCTOSE/MIN/mg PROTEIN

0.9

0.6

0.3

 

L-RHAMNOSE
_._Mn++
_o_ CO'H'

— \M

No Metal

 

LmMANNOSE

No Metal

—_———-—_—*——

 

I -l l l- l I

 

o 10“"1 10“”2 10*} 10““ 10"“5 10’0

CONCENTRATION OF METAL ION (M)

83

 

TABLE VII
++ ++ L. ..
Effect of Mn and Co on tne isomerization of L-rhamnose and Lomannose
The standard isomerase assay was employed. The cells were grown
on L-rhamnose and the crude extracts were used. The assay concentrations
++ ,

++ '3 , . .
for Mn and Co were 5.4 x 10 M and increases in the concentrations

of each cation were made acccrding to the desired ratio.

 

 

 

Substrate Metal Ions Specific Activity
(Ratio) pmoles ketose/min/mg protein
Ithamnose none 0.hh
Mn++ 0.60
Co++ 0.29
Cd++JMn++(l:1) 0.25
Co++3Mnf+(1:2) 0.2M
Co++iMn++(lz5) 0.25
L-Mannose none . 0 . 25
Man—+ 0.08
Co++ o. M.
cd++gnfi++(1 l) 0.27
Co++zMnf+C5 l) 0.56
CO++2Mn++(1;2) 0.2L
Cof+zMnf+(1:h) 0.1M

 

85

TABLE VIII

Effect of mixing substrates on isomerase activity

 

The Roe Test (12) was employed to determine activity in crude

extracts on Lomannose and mixtures of L-rhamnose and anannose.

The concentration of each substrate was the same as described in

the routine assay except that no metal ions were

included.

 

 

Substrate

0.D. 2O/20 min

 

 

 

 

5
LmMannose 0.290
L~Rhamnose 0.085
LAMannose + L-Rhamnose 0.070
(noC:::;;:§ Sample (umoles) O'D°520
LoFructose 0.25 0.550
L-Rhamnulose 0.50 0.050
L-Rhamnose 0.50 0.015
L-Fructose + Lu-Rhamnose 0.25 + 0.30 0.31.5
L-Fructose-+ Lthamnulose 0.25 + 0.50 0.590

 

 

 

86

.uomuuxo HHwo oSu mo coauowum mummasm sowsoEEm so an zufl>fiuo<*

.aflououm mo wE\cHE\uoscoum mo moHoai mm commoumxo mum mufizmmm

 

 

 

 

 

H0.0 no.0 Hm.o O®.O HO0.0V HO0.0V wm0c¢m2nA
AquuDEv
No.0 no.0 OM.H OH.~ HO0.0V HO0.0V omocfimnmuu Nmum
mo.o mo.o om.H OH.H mo.o HH.O mmocnm21H
Ausmummv
no.0 HH.o o:.H mm.H no.0 sm.o mmeaemam-a masque
m-H- m-H-
omouusumaa omoHJcEmAmuA mmouonumuu mmoHsnamnm-A omcssz-A mmonEm£MuA HmosncH cflmnum
immmaocfi< *mmmnflm mmmnofiomH

 

 

 

 

.uoumm>umn ohms
mHHmo on“ mouzcaE on we oswu toquuoccfi cm swumm com coaumfisoczfl w¢umm meme; 0 cocoa mm3 omo:Emnm
IA .nzmop cm; nonummflfiaun mmonCmfi-A cons woumw>umn wum3 mflfloo ago wow scmumanuocfl mum3 mHHwo «no

mafia 08mm msu um Bahama ausoum can we cwocm mm: mmoncmzAH .Anomw ma Ommv mmochEuA no wmonEwnH

IA uwfiuflm Sues couswEmemsm Luopn unwfluuoa mo HE 00m 5H nachm onwz mHHmo mmum cam mmhu cHHS

 

 

unmasa o>Hunwoszomommmnkug\omonomaaq m com “mm-ammv damn cHfi3 mo muomnumw ow mowufi>fiuom maNNmm

xH mqm<fi

87

was not indiced by either Lamannose or Iwrhamnose, and
no isomerase activity was d--ected on either of these
two substrates.

(9) pH Optimum— The pH Optimum of the isomerase

 

was determined with both L-mannose and Lwrhamnose as

substrates and in the presence and absence of their

activating metals (Figure 24). In the presence of cd*+.

Lemannose was isomerized maximally over a pH range of

4.

7.5-7.7, and Without Co‘ over a pH range of 7.4-8.5.
The decrease in the pH Optimum in the presence of Cc>++
probably was due to cobalt hydroxide formation.

The shift in the pH Optimum.was more clearly defined

when L-rhamnose was the substrate. In the presence of

++

Mn L-rhamnose was isomerized at a maximum rate when

3

, ++ .
the pH was 7.3-707 and in the absence of Mn a pH shift
to 8.7-9.0 occurred. The rapid decrease in L-rhamnose
isomerization after pH 7.? probably was due to manganese

dioxide formation.

(h) Temperattre Ootimsm- With Iwrhamnose as the

 

substrate and Mnfl+ as the activating metal, the isomerase
exhibited a temperature Optimum of 60-650 compared to

. +4." . .
55—600 in the absence of.Mn (Figure 25). With L—mannose
as substrate the isomerase in the presence or absence of

Coh+ had a temperature Optimum of 50-550.

Fig. 24. The pH Optimum of the isomerase. The stan-
dard assay for measuring isomerase activity was
employed. The crude extract was fractionated on
Sephadex G~lOO and the fraction having the highest
isomerase activity was used. The amount of protein
per assay was 0.52 mg. Each of the buffers employed
was 0.02 M and had the following pH ranges: sodium
phoSphate (pH 6.7-7.5), tris-HCl (pH 7.4-8.8), gly-
cine-NaOH (pH 8.9-9.4)9 and carbonate-bicarbonate

(pH 9.8-lO.l). The pH of the reaction was record-d

at the end of the incubation period.

umoles LuFBUCTOSE/BO MIN

umoles L-RHAMNULOSE/lo MIN

Figure

1.5

0.5

1.5

0.5

24.

 

LmMANNOSE

._ CO++

 

o— No Metal

e/o’o—O—L
a 1

 

’.L~RHAMNOSE

Q_.Mn++

O-No Metal

 

 

 

7.0 8.0 9.0 10.0
pH
89

90

Fig. 25. Temperature Optimum of the isomerase. The
enzyme was fractionated on Sephadex GelOO before use.
To measure isomerase a-tivity the assay mixture con-
taining all the components except the substrate was
heated for five inates at a Specific temperature,
after which the substrate was added to initiate the
reaction. When Lwrhamnose was the snbstrate, the
assay contained 0.17 mg of protein; when meannose
was the substrate, the reaction mixture contained

0.52 mg of protein.

Figure 25.

Aoov mmseammmsme

om on om

om

0: on

 

 

_ fl _

Hmpmz oz «0

++oo :0

_

 

Mmozz<21A

 

 

o.m

0.:

MIN OC/HSOLOflHfl‘T setomn

Aoov mmseammmzmea
om on om on 0:. on

 

 

_ _ _

   
    

Haves 02 IO

++sz I o

mmozz¢mmlq

 

 

0H

NH

ad

0H

NIH OT/HSOTHNNVHH”T setomfi

91

92

Properties of LoFructose (L-Rhamnulose) Kinase:

(a) Activity of the Kinase in Crude Extracts- The
rates of phOSphorylation of L-fructose and L-rhamnulose
were compared as a ftnction of kinase concentration in
crude extracts of both wild-type and mutant cells (Figure
26). Although the kinase activity varied in each extract.
the ratio of the activities of L-fructose to L-rhamnulose
remained constant.

(b) Partial Purification of the Kinase- The kinase
was purified about 7—fold with a 20% recovery of activity
(Table X). Fractionation with ammonium sulfate and Sepha-
dex G-100 failed to separate the L-fructose from the
L—rhamnulose kinase activity (Figure 27).

(c) Km of LmFructose (L-RhamnuloselKinase- The Km's
for both L-fructose and Lmrhamnulose were the same for
partially purified kinase obtained from both wild type and
mutant cells (Figures 28 and 29). The kinase bound
Lmrhamnulose more strongly than L~fructose. The Km value
for L-rhamnulose was 0.05 mM compared to 1.7 mM for L-
fructose.

(d) Substrate Specificitye The kinase was Specific
for L-fructose and Lmrhamnulose (Table XI). Activity on.
D-fructose was attributed to D-fructose kinase.

(e) Effect of Mixing Substrates- Kinase activity

on L-fructose and Lmrhamnose was not additive (Table XII)

- uulfii“. ll. . m:
“Mb
|

.v'

III“~ r‘m‘Irb‘

93

Fig. 26. Activity of L—fructose (L-rhamnulose) kinase
in crude extracts. The standard kinase assay was

employed except for the change in protein concentration.

Figure 26 .

szBomm ma

3.0 m.o N.o Hoo

 

 

omoassamnmaq Z

 

7 mmopogmnq

BZdHDS

  
  

 

om o.Q:.§.m£maaH\v

4f!

omopofihmaq

mmHB QHHz

 

 

on

or

om

NIH/deVEmS setomu

94

S
O/

.muzawa nod pmumamuosamoad macasdswsuuq no mmouosumng mo mmaoaouowz.*

 

Hm.m mm.m
ww.o Hw.o
hm.o Om.o

macaadamsuua omou03HMuA

 

 

afimuoum‘ a\muwcb

Hm
ovumw

OOH

 

:H ma
m: mm
mm as
macassamnuua mmouosumnq
Ll.
*muHGD

ooH-o xmemsamm
mummasm aswaoaa€

uumuuxm dunno

 

sua>auu< oawdumam

zum>oomm

mufi>wuu< Hmuoe

coauomum

 

 

 

mHHmu omhunpaws scum ommaax AeneasdEmnuulq‘mmouusuwua mo dowumowmausm Hmfiuumm

N mam<H

1 .

96

Fig. 27. Fractionation of the kinase on Sephadex
G-100. Details of the procedure are in the text.
Each fraction was assayed for kinase activity on

L-fructose and L-rhamnulose.

mg PROTEIN/FRACTION

Figure 27 .

 

30 __

 

MUTANT

o->Protein
.— L-Fructose
9- L-Bhamulose

 

 

WILD TYPE

__ 60

 

 

 

 

FRACTION NO.
97

nmoles ADP/5 MIN

“'1

l

1rillrt.fv\ vne,

'

F.
a.
v

98

Fig. 28. Lineweaver-Burk plot of L-fructose (L-
rhamnulose) kinase from L-mannose-positive cells.
The standard kinase assay was used except that the
substrate concentration was varied as indicated with

the kinase (Sephadex G—100 fraction) concentration

constant.

Figure 28.

 

Km for L-Fructose =
1.60 x 10-3M

 

 

 

 

 

L-Fructose (mm)

 

for L-Bhamnulose =
4.3 x 10-5M

.l x 10”3
v

0.5

 

 

 

' I l l l l

 

 

o 0.1 0.2 0.3 0.4 0.5

1
L-Bhamnulose (mm)

99

.eui

I.‘ L‘w‘ .

100

Fig. 29. Lineweaver—Burk plot of L—fructose (L-

rhamnulose) kinase from wild-type cells.

Figure 29 .

 

 

 

 

 

 

 

 

 

Km for L-Fructose =
1.67 x 10‘3M
2.0...
3]? x 10'"3
1.0__
/ I I I I I
o 1 2 3 4 5
___A 1
L-Fructose (mMT
Km for L-Rhamnulose =
5.2 x 10-5M
1.0—
% x 10"3
0.05-——
l J l l I
0 001 002 003 001* 0.5

1
L-Bhamnulose (mMT

101

 

 

102

TABLE XI

Substrate specificity of the kinase

 

The routine kinase assay was employed. With the exception
of L-rhamnulose the assay contained 1.5 pmoles of each substrate.

The enzyme was fractionated on Sephadex G-100 before use.

 

 

 

 

 

 

L-Rhamnose-Grown Cells LdMannose—Grown Cells
Substrate Comparative Rate (%) Comparative Rate (fi)
L-Rhamulose 100 100
L-Fructose 110 110
D-Fructose 1.8 2.5
L-Sorbose 0 0

L4Mannose 0 0

 

105

TABLE XII
Effect of mixing substrates on kinase activity
The routine kinase assay was used. 'When both substrates
were mixed the concentration of each substrate was the same as
that used in the routine assay. The kinase was fractionated

on Sephadex G-100 before use.

 

Growth Substrate

 

Substrate L-Rhamnose L4Mannose

 

 

 

Specific Activity (pmoleslmin/mg)

L-Rhamnulose 1.76 1.h3
L-Fructose 1.95 1.60

L-Rhamnulose-+ L-Fructose 1.85 1.55

 

 

 

104

when assayed from cells grown on either L—mannose or
L-rhamnose.

(f) pH Optimum- The pH Optimum of the kinase ranged
from pH 7.0-8.0 in both wild—type and mutant cells when
either L-fructose or L-rhamnulose was the substrate (Fig-
urea 30 and 31).

PrOpertie; of L-Fructose-l:g_(L-Rhamnulose-l-P)

Aldolase:

 

(a) Activity in Crude Extracts— The rates of cleavage
of L-fructose-l-P and Lprhamnulose-l-P were compared as
a function of aldolase concentration in crude extracts
from both wild-type and mutant cells (Figure 32).

(b) Partial Purification of the Aldolase- The aldo-
lase was purified about 6—fold with a 20% recovery of
total units (Table XIII). Fractionation with ammonium
sulfate and Sephadex G-100 failed to separate the L-
fructose-l-P from the L-rhamnulose-l-P aldolase activity
(Figure 33).

(c) Km of the Aldolase- The aldolase bound Lprhamnu-
lose-l-P 10 times more strongly than Lpfructose-l-P.

The Km value for L-rhamnulose-l-P was 0.5 mM compared to
5 mM for L-fructose-l-P (Figure 34).

(d) Effect of Mixing Substrates- ‘When L-fructose-l—P

and Lprhamnulose-l-P were mixed together, their combined

 

l‘ ne'er."

105

Fig. 30. pH Optimum of the kinase in wild-type cells.
The routine kinase assay was used. The kinase was
fractionated on Sephadex G-100 before use. Details
of the buffers and pH ranges used are given in Figure
24. The pH of the reaction was recorded at the end
of the incubation period. The amount of protein per

assay was 0.08 mg.

Figure 30.

 

 

 

 

 

 

L-RHAMNULOSE
20...
-0- Tris-H01
10 _ —O — Ph08phate
a -I - Glycine-NaOH
”Q
a I - . l I I
3 L-FHUCTOSE
H
E
30.__
20.__
(M
10,——
I I I I
6 7 8 9

pH
106

107

 

Fig. 31. pH Optimum of the kinase in the Lemannose-
positive cells. Refer to Figure 30 for details. The

amount of protein per assay was 0.04 mg.

Figure 31.

 

30..—

20...

 

10 ___ -O-—Tris.HCl
-9 -PhOSPhate

 

 

a - I - Glyc ins-NaOH
2
Q _-
a l A f I l I
m L-FBUCTOSE‘
.9.
E3
30 __.
20 —
l 0 _

 

F—
—

 

pH

108

 

109

“E

tans”— "

Fig. 32. Activity of L-fructose—l-P (L-rhamnulose-
l-P) in crude extracts. The routine aldolase assay

was used except that the protein was varied.

Figure 32.

 

szeomm ms
30.0 «0.0 o

     

mIHIomoposHmlq

.\\IJW
mIHImmOHsssdsmlq

NAME QAHz

 

30.0 No.0

   

mIHIomopoohmIA

mnflummoasnamsmuq

BZdBD:

 

 

NIH/JVHG satomu

110

111

.zmmmm pumuamum msu cw 0mm um munowa Hon pouflpaxo mm<z mo mOHOEonOHZ *

 

 

 

 

Rd S .o E Tm To 02 -o xoemaamm
mm.0 0H.0 ow m.m :.m ouwmaom Esadoefi<
No.0 no.0 00H 0.HH 0.: uomuuxm dunno
mIHuwmoHsnEmnuuq muanowouusumuq mIHIOmoHsoEmsuIA mnauwmouusung
rll |
cwououm wE\muHo: fl *muadb
aufi>wuu< oamaommm zum>oomm zua>wuo< kuOH dowuomnm

 

 

 

udmusfi w>fiuamo Immoodwaua map Eoum wmmaovam

 

 

 

NmIH-mmoHscsmnuuav muaummouoonwng mo dofiumoamwus Hmauumm

HHHN mam¢a

112

 

Fig. 33. Fractionation of the aldolase on Sephadex
G-100. The routine aldolase assay was employed.
Details of the procedure are outlined in the text.
Each fraction was assayed for activity on L-fructose-

l-P and L-rhamnulose-l—P.

Figure 33 .

mg PROTEIN/FRACT ION

 

 

 

 

MUTANT
16" o-Prote1n "" 4
o- L—Fructose-l-P
111__ o- L-Rhamnulose-l -P
12 —- —. 3
O
10.—
8 L_ _ 2
6_ '
.\
1+... ’ \ ° ._. 1
\\ O
O
2__ \\ .
\ O
‘O\ o
I I I \
0 LI 5 6 7

 

FRACTION NO .

113

pmoles DHAP PRODUCED/MIN/FRACTION

114

Fig. 34. Lineweaver-Burk plot relating aldolase
reaction velocity to substrate concentration. The
routine assay was used except that the substrate
was varied as indicated with the aldolase (Sephadex
G-100 fraction) concentration constant. The enzyme

was obtained from cells previously grown on L-mannose.

Figure 34.

 

Km for L-Bhamnulose-l-P =
5.0 x 10'

 

 

 

 

 

 

 

 

 

 

 

/ I I I
0 0.1 .0.2 0.3
.‘ . 1
L-Rhamnulose-l-P (mm)
Km for Fructose-l-P =
5.0 x 10‘3M
8.0.——
6.0....
l x 1.0"3
4.0——
2.0 —"
I I I
0 2 4 6

L-Fructose Imfi)

115

116

aldolase activities were not additive (Table XIV).

(e) Substrate Specifigityr The aldolase when
purified from both.wild-type and mutant cells had the same
substrate Specificity (Table XV). It cleaved only L-
rhamnulose-l-P and L—fructose-l-P, and did not cleave
D-fructose-l-P or D-glucose-l-P. Activity on the other
substrates was due to contaminating enzymes in the
preparation and are listed in the table.

DISCUSSION

The evidence Obtained in Part II of this thesis
indicates that the same enzymes degrade L-mannose and
L—rhamnose (Figure 35). They were induced by Lemannose
and Lnrhamnose in both the‘wildatype and mutant cells and
the ratios of the Specific activities of the individual
enzymes for Lemannose, L-rhamnose, and their metabolic
intermediates were the same in each strain. In addition,
whether the enzymes were isolated from the wild-type or
mutant cells, the activities on Lemannose or L-rhamnose
could not be separated when subjected to partial purifi-
cation. The enzymes showed the same substrate specificity
when induced by either Lphexose and their individual
Specific activities were not additive when measured in -
the presence of both substrates. Finally, neither L-

mannose nor Lprhamnose was able to induce the isomerase

117

TABLE XIV
Effect of mixing substrates on aldolase activity
The routine aldolase assay was employed. When both sub-
strates were mixed, the concentration of each substrate was the
same as that used in the routine assay. The aldolase was frac-

tionated previously on Sephadex G-100 before use.

 

 

Substrate Specific Activity (pmoles DHAP produced/
min/mg protein)

 

L-Fructose-l-PO4 0.20
L-Rhamnulose-l-P04 0.33

L-Fructose-l-P04 +
L-Rhamnulose-l-P04 0.24

 

118

TABLE XV

Substrate specificity of the aldolase

The routine aldolase assay was used. With the exception of L-

rhamnulose-l-P, the assay contained 1.5 nmoles of each substrate

when tested. Contaminating activities are listed in cases where

activity was expressed but was not due to cleavage by the aldolase.

 

 

 

Substrate Comparative Rate (%)
L-Rhamnulose-l-P 100
L-Fructose-l-P h0-60
D-Fructose-l-P 0
D-Glucose-l-P 0
D-Fructose-6-P mannitol-l-P dehydrogenase activity
D-Glucose-6-P D-Fructose-6-P isomerase activity

D-Fructose-lr, 6-diP

glycolytic aldolase (trace)

 

 

Figure 35.

mmwmmmfldfioddlq

madmmmomm
mzoamodeommfimHm
mmwmmqqdmmquclg

madmmwcmm
MZOBHU¢HXOmQHmHQ

 

mIHImmOADzzdmmIA Alllll. mmOADzz<mmlq
mad

mlalmmOEODmhfq Alllllu mmoaoammlq
mad .

mmozzdmmifl

HWOZZdSIA

119

120

in a mutant deficient in this enzyme.

The metabolism of L—rhamnose has been described
previously (36-41) and also found to undergo isomerization
to L-rhamnulose, phosPhorylation‘with.AT? to L-rhamnulose-
l—P, and cleavage to dihydroxyacetone phosPhate and L-
lactaldehyde. Domagk and Zech (37) purified L-rhamnose
isomerase from Lactobacillg§_plantarum and showed that it
isomerized Lemannose at a rate of'<l% that of L—rhamnose.
Chiu and Feingold (67) reported that L—rhamnulose-l-P
aldolase from g, ggl;_would not condense dihydroxyacetone
phosphate and L-glyceraldehyde but Ghalambor and Heath
(43) showed that L-fuculose-l-P aldolase would condense
them to yield L-sorbose-l-P. In most cases, however,
L-mannose or its metabolic intermediates have.not been
employed as test substrates.

The mutual antagonistic effect of Mnh* and Co++ on
L—mannose and L-rhamnose is an interesting characteristic
of the isomerase. Swada (39) showed that L-rhamnose
isomerase from Q, ggli_was stimulated by Mnh+ and inhibited
by Coh+, but L—mannose was not tested as a substrate.
Similar cases of changes in substrate Specificity due to

changes in cations have been reported. Folk and Gladmer

(68) showed that Co*+ and CdF+ produced changes in the

121

peptidase and esterase activities of carboxypeptidase B.
CoF+ increased the peptidase activity and inhibited the
esterase activity whereas Cd++ reversed the phenomenon.
DNA polymerase also shows a similar effect (69); normally,
the reaction proceeds in the presence of Mg*+, and only
deoxyribonucleotide triphOSphates serve as substrates.
If Mn++ replaces Mg*+, however, ribonucleotides also may
serve as substrates.

D-Mannose isomerase from Pseudomonas saccharophilia
(70) has been purified about 6-fold and shown to be
highly specific for D-mannose ande-rhamnose but inactive
on Lemannose and Lprhamnose. DeMannose isomerase, unlike

Lsmannose isomerase, is not affected by MnF+ or Co++.

 

PART III
Comparison of the Utilization of L~Mannose by

:Wild-Type.§, Aerogenes and the LeMannose—Positive Mutant

The first two parts of this thesis defined the bio—
degradative pathway of Lemannose in a, aerogenes and
confirmed that Irmannose and L—rhamnose were metabolized
by the same enzymes. A mutant strain of A, aerogenes
has been isolated which, unlike the wild type, readily
grows on Lsmannose as the sole carbon source. The third’
part of this thesis investigates the basis for the gain
in the ability of this mutant to grow on Lemannose.

EXPERIMENTAL PROCEDURE

growth Curves- Growth curves were performed in
test tubes (150 mm x 18 mm) containing 7.0 ml of mineral
medium or nutrient broth (71). The mineral medium was
supplemented with 0.28% hexose andinoculated with 0.05
ml of actively growing cells to give an initial O‘D’540=
0.01. (For growth recorded on limiting amounts of hexose,
the mineral medium or nutrient broth was supplemented with
0.08% hexose solution. The tubes were shaken at 30°
on a reciprocating shaker. All growth measurements were
recorded at 540 nm.with a Coleman Junior SpectroPhotometer.

Analytical Procedures— L-Rhamnose and Lemannose -

were determined by the method of Folin and Malmrose (11).

122

 

123

When the L—hexoses were mixed together, L—rhamnose was
determined by the cysteine-sulfuric acid test (72) and
L-mannose determined by the difference in the total
reducing sugar present.

Reagents- LrGalactose was prepared by the method

of Frush and Isbell (73) (courtesy of R. Hart). DL-

Glyceraldehyde was purchased from Calbiochem., Los Angeles,

California. Chloramphenicol was purchased from the
Parke Davis and Company, Detroit, Michigan, and puro-
mycin was a gift from Dr. A. Morris.

Determination of the Number of Generations- The
total number of cells during growth was determined with
a Petroff-Hauser Counter and viable cells determined by
plating on nutrient agar.

RESULTS .

growth of‘Wilg;gype Cells on D:§lucose. L;Bhamnose,
and LeMannose- 'Wild-type A, aerogenes readily grew on
L-rhamnose but no significant growth occurred on L-mannose
during the same incubation period (Figure 36).

Selectign of the L-Mannose-Pogitive Cellgf If the
wild-type cells were incubated with agitation for an
additional period of time with L-mannose, growth on L—
mannose eventually occurred but at a very slow rate.

If then these cellsvmna inoculated into fresh mineral

124

Fig. 36. Growth of wild-type cells on D-glucose,

L-rhamnose, and L-mannose.

Figure 36.

 

 

 

 

 

 

l l l I
008 F. . . . . . .—
O
0.6 __ o— D-Glucose
g .- L-Rhamnose
Q1“ . g- L-Mannose
:5
0.4 _ __
O
0.2 h— '——l
O
~L
5"; . ‘1' D J—1 4' {F1 1 fl
0 5 10 15 20
HOURS

125

126

medium supplemented with L—mannose, growth occurred at a
faster rate and by subsequent transfer a strain was
eventually selected which readily grew on Lsmannose.
Figure37 shows the relation between generation time and
transfer number. There was a gradual decrease in the
generation time to a minimum of 2.5 hours.

Stability of the L—Mannose-ngitive Mutant- The L-
mannose-positive strain retained its ability to grow on
L-mannose. The cells could be stored for several weeks
on nutrient agar and when inoculated into medium supple-
mented with L-mannose, the cells grew readily. Further—
more, if the mutant cells were grown on other carbon
sources, such as D-glucose or L-rhamnose, they retained
their ability to grow on Lemannose (Figure 38), indicating
that growth on L-mannose was due to a mutation rather
than an adaptation.

Response of the Wild Type and Mutant to Imvic Tests
and a Specific Phage- Both wild—type and mutant cells
resPonded identically to Imvic tests (71) (Table XVI)
and to a phage which lysed A, aerogenes (Figure 39),
indicating that the L-mannose positive cells selected
were derived from.a, aerogenes and not a contaminant.

Diauxie Curygs—ggffect of_;gMannose on the Growth

of the Wild Type and Mutant- Since the manometric studies

127

Fig. 37. Selection of the L-mannose-positive mutant.
Each transfer was performed after the growth culture
reached an O'D'540 = 0.40. A 0.05-ml volume of cells
was transferred to fresh medium supplemented with
L-mannose. The generation time was defined as the
time in which the culture grew from an O.D. =

540
0.15 to 0.30.

Figure 37 .

 

 

 

 

 

_
0
3

_ _
0 0
2 1

AmmDOmv mzHB 20H “magma

12 ll» 16

10

TRANSFER N0 .

128

129

Fig. 38. Stability of the Lsmannose-positive mutant.

 

Figure 38.

 

 

mmoom
mm om mg as n o
O
C
O
0 III No0
.
a ll #.0
0
I1 000
.o
C O C m o
omosadzmiq so whommndha
mouse sound omozsmzlq no 593090 to mbam
omossmzaq so Spsoho no

 

omoosHUIQ so mummmsmna
your: omogznq so spZOHQ IO,

omosstIA no szoso.uo

 

N.o

3.0

0.0

m.o

 

OthCIoo

130

131

TABLE XVI
Response of the wild type and mutant to imvic tests
The tests were performed according to the procedures outlined

in the Difco manual (71).

 

 

J_

 

Cell Type Indole Methyl Red Voges-Proskauer Citrate
Wild + + - +
LéMannose

Positive + + - +

 

Fig. 39.

132

Phage Specificity of A, aergggges.

_‘_._-_——'«-—.——— -

Figure 39.

 

 

BZdBDz

 

mmDom,

N6

0.0

s
a

 

mmwe QAHz

 

N.o

3.0

0.0

009.0.0

133

134

presented in Part II showed that the wild-type cells
could utilize L-mannose if the biodegradative enzymes
were previously induced by L-rhamnose, investigations
were carried out to determine what effect L-mannose had
on actively growing wild-type cells. Since the wild-
type cells grew very slowly on L-mannose as a sole carbon
source, the growth medium was supplemented with a limiting
amount of L-rhamnose to induce the degradative enzymes,
and, after the L-rhamnose was completely metabolized,
Irmannose utilization would begin. Figure 40 compares
the utilization of L-rhamnose and Lsmannose by wild-type
cells. The cells grew preferentially on D-glucose, and
when the D-glucose was completely degraded, they grew

on L-rhamnose but not on L-mannose. L-Mannose-positive
cells produced a different result (Figure 41). D-Glucose
and L-rhamnose were readily utilized but, unlike the

wild type, the mutant cells also grew on Lemannose.

When the wild-type cells were allowed to grow on a
mixture of L-mannose and L-rhamnose, a change in the growth
rate occurred; growth on L-rhamnose was rapid and when
completely metabolized growth on Limannose began but with
a decrease in the growth rate (Figure 42). In contrast,
the mutant readily grew on L-rhamnose and L-mannose, and

in the mixture of these hexoses the cells grew to a greater

 

135

Fig. 40. Growth of wild-type A, aerogenes on D-
glucose, and mixtures of D-glucose and L-rhamnose,

and D-glucose and L-mannose.

 

 

0H 0 0
I _
all. N00
[:00
mmozz<zlq
+ mmocbnwlm
L000

Figure 40.

OH

mmDom

mmozzdmmlq
+ mmOODAGIQ

 

~10

0.0

 

 

OH m o
_ _
[NCO
06
o 1/
G1
c“
h.
o
[:00
mmOODHUIQ
[0.0

137

Fig. 41. Growth of the L-mannose-positive strain
on D-glucose and mixtures of D-glucose and L-rhamnose,

and D-glucose and L-mannose.

 

 

 

 

 

 

 

1... m6
mmozzazuq
+ $830.0 . mmozzqmmuq
I: 0 o + 8830-0 1 .
o o WmOUDAOIQ 10.0

Figure 41.

138

04790.0

139

Fig. 42. Growth of the wild-type cells on L-rhamnose,

L-mannose, and a mixture of these L-hexoses.

 

 

mmozzazlq
+ mmozz<mmlq

 

Figure 42 .

:.0

0.0

ON

mmDom

OH

 

mmozz¢21A

 

N.o

ON

OH

 

mmozzdmmiq

 

No0
0%
G1
r...
h.
0
14.0
0.0

141

extent than the wild type with no change in the growth
rate when Lsmannose utilization had begun (Figure 43).
Thus, when L-mannose entered the wild-type cell, L-
mannose itself, or its metabolic intermediates, exerted
a toxic or inhibitory effect on the organism. This
effect was overcome by the mutant resulting in active
growth on L-mannose.

Hexose Utilization as a Function o§_Growth- To
demonstrate a more direct effect of Lsmannose on the
growth of wild-type and mutant cells, growth and hexose
utilization were measured simultaneously. Figure 44 com-
pares the utilization of L-rhamnose and Irmannose as a
function of growth. L-Rhamnose, but not Lemannose, was
readily utilized by the wild type. When both hexoses were
mixed, the wild-type cells showed a preferential growth
on L-rhamnose which, after completely degrading it, began
to metabolize L-mannose. However, the growth rate on
Lemannose decreased, eventually leveling off but with
continuous utilization of L-mannose. Unlike the wild
type, the mutant readily grew on L-mannose after the L-
rhamnose was metabolized (Figure 45).

Although the diauxie studies indicated that L-mannose
exerted an inhibitory effect on the growth of the wild-type

cells, these investigations were conducted under conditions

 

142

Fig. 43. Growth of the L-mannose-positive strain
on L-rhamnose, L-mannose, and a mixture of these

L-hexoses.

 

Figure 43.

 

mmOZZ¢zIA
+ mmozz¢mmlfl

 

N.o

:.0

ON

.mmDom

0H

mm02242|u

 

N.o

:5

 

mmozz<NMIA

 

N.0

:.0

0.0

mam,

143

144

Fig. 44. Determination of Lemannose and L-rhamnose

utilization by actively growing wild-type cells. Hexose

utilization was stOpped by removing 100 pl aliquots

from the growth medium and adding it to 100 pl of

0.2 N H2304. Aliquots of this solution then.were

tested for hexoses by the following procedures:
a. 'When either L-rhamnose or L-mannose was the
sole carbon source, it was measured by the method
of Folin and Malmrose (11).
b. -When L-rhamnose and Lamannose were mixed,
L-rhamnose was measured by the cysteine-sulfuric
acid test (62). The difference in Optical den-
sity at 396 nm and 429 nm gave the amount of L-
rhamnose in the presence of meannose.
c. To determine L-mannose in the presence of
L—rhamnose, the total optical density of the
mixture was determined by the procedure of Folin
and Malmrose. With L—rhamnose determined by the
procedure in part b, its contribution to the Opti-
cal density measured at 520 nm in the mixture was
calculated. Thus, the difference in the Optical
density of the mixture and the calculated value

for L-rhamnose gave the amount of L-mannose.

145

 

 

 

(Wm) NOIIVHLNHONOO asoxaa-q

Figure 44.

 

mmoom
0m 0H 0
nYIIlAYMflWATIIIAYIAU
spacso
m1: .1 m.o
:1! all omossmzwq II 0.0 mmosamsmn
0|! 01! IL 0.0

 

 

 

 

 

 

 

 

Ofig-q-o

146

Fig. 45. Utilization of L-rhamnose, Lemannose,
and a mixture of these L-hexoses by actively growing
L-mannose-positive cells. Refer to Figure 44 for

details.

(Mm) NOImvamNsonoo HSOXHH'T

Figure 45.

 

 

 

 

m mDom

 

      

0N 0H 0

/

nnzoso

 

 

0| [0.0

OH.

147

 

 

  

7:380

\,

mmosSQQmsq

   
  

 

148

in which the biodegradative enzymes were induced by

L-rhamnose. Both cell types reaponded differently

 

to growth on nutrient broth supplemented with L-
mannose (Figure 46). The growth of the parent cells
was inhibited when utilization of Lemannose had begun
but with continued uptake of L-mannose. Likewise, the

mutant cells were inhibited when L-mannose initially

 

was utilized, but unlike the wild type, the inhibition
'was overcome and growth on Lsmannose continued to com—
”pletion.
Ipduction of L-Mannose Degradative Enzymesgigithe
(Wild Type- The isomerase, kinase, and aldolase acti-
vities were measured in the wild-type cells after induc-
tion by Lemannose and L-rhamnose (Table XVII). Both L-
hexoses induced all three enzymes which explains the
utilization of Lamannose by the wild-type organism.
Measurement of Enzymatic Activity upon Removal of
th§_1ndug§;r To demonstrate if any or all of the first
three degradative enzymes were constitutive in the L-
mannose-positive cells, the inducer, L-mannose, was
removed and the cells were grown for four and seven
generations (Table XVIII) after which individual enzyme

activities were measured (Table XIX). The simultaneous

149

'Fig. 46. Growth of wild-type and L-mannose-positive

cells in nutrient broth supplemented with L-mannose.

Figure 46.
0.8

0.6

0.4

0.2

O
:1-
Y‘ o
9
O
0.6
0.4

0.2

 

 

 

 

 

150

WILD TYPE
__8
O
L-Mannose.‘_____;7
__6
O
. O
’ o
. o 0...“,
0
Growth
k/
' ——2
O
' I 1 1 o
L-MANNOSE POSITIVE MUTANT
. O
__ o __6
L-Mannosec§__,;7 '
O
0 -—Lb
o 0 ’
O
O
O
. Growth
<—/ .-
. 7
. l J l
3 '6 9 12
HOURS

L-MANNOSE CONCENTRATION (mM)

 

151

.cfiououm ma\o«a\uospoum moHosi mm vommoumxo www.mmwuw>wuo<

.oummaom soaaoaam nuHB woaumGOwuomum Houwo poawsuouop whoa moaua>wuom ommfiopam 0am mmmcax 0:9 *

 

 

 

00.0 00.0 0m.s 0H.H 00.0 HH.0 mmoaamzwa
no.0 HH.0 0:.H mm.a s0.0 sm.0 mmoasmam-a
m-~- m-H- 44
mmouosumuu omoHssamAMuA omouosumua anodsssmamug omonsszA omOnEM£MuA
*ommaopa< *mmmaaM mmmumEOmH HoosocH

 

 

 

 

 

. .umo>um£ muomon massage Oh Hmcoauwpvm

cm 0x030 Ou posoflam paw omosamnuug mo we 0mm :uHB pmudmswamdsm mums moan aoasa umumm «mason
0-0 you :uoun usoauus: as mHHonnouom saouw whoa mHHoo 0:9 ummossonMuA kn sowuospsH .n

.0oosoomu

was omoaomsiu an nuaouw mo cowufinwncw cos? woumo>umn one: mHHmo 0:9 .mmosamsrA mo wa_0mn nuwz

pouaoEwHQQSm suoun uswwuusc cw zaamownouom aaouw mums mHHwo may nomoaaszq mp coauusoaH .m

 

mHHoo unauupaga aw moshuso omossmaiu mo aoauospoH

HH>x man<a

Determining the number of‘generations

152

TABLE‘XVIII

upon removal of the inducer

A culture of the mutant cells previously grown on Ldmannose was

diluted to a final O.D.

54o
ed into 490 m1 of nutrient broth and allowed to grow.

= 0.50 and 10 ml of this solution inoculat-

The total

number of cells at a specific time interval was determined with a

Petroff-Hauser bacteria counter and the viable count determined by

plating the cells on nutrient agar.

This procedure was used to

determine when the cells had grown to four generations. For seven

generations of growth, 2 ml of the diluted mutant culture was inocu-

lated into 498 ml of nutrient broth and the above procedure repeated.

 

 

Four Generations

 

 

 

 

 

 

Hours of Growth O.D. Viable Count Total Count
540 (cells/ml) x 108 (cells/m1) x 108

0 0.010 0.21 0.20
1.25 0.016 0.25 0.25
2.00 0.038 0.30 0.40
2.75 0.080 0.50 0.56
5.50 0.168 1.55 1.40
4.75 0.500 5.50 4.60

Seven Generations

0 0.001 0.05 0.05
1.25 - 0.05 0.08
2.00 - 0.07 0.10
2.75 0.025 0.15 0.20
3.50 0.055 0.28 0.38
4.25 0.140 0.65 0.80
5.00 0.290 3.30 1.94
5.75 0.400 5.70 4.00

 

153

N00.0 V *

.aamuoud mo wa\awa\uo=0oum mo moHoai mm vommmumxm mum muHsmmm

 

*0

*0

00.0

*0

*0

0H.o

*0

m00.0

an.0

*0

moo.0

hm.o

*0

m00.0

0N.0

*0

000.0

00.0

suoum
unmauusz

aboum
usoauuaz

mmossszA

 

man -
mmouosumug

muHu
omoHsnsmAMuA

 

omouosumufl

 

mmoHssawnMuA

«woodmanfi

 

mmoasmAMuA

 

006.9 OUH<

 

«madam

 

ammumsOmH

 

msofiumumsou

 

oumuumnsw
nusouo

 

 

.uxmu was :« vmnauomov mm wwuommoa muomuuxw musuo mSu a“

xuupwuom oshusm 0cm 00u00>um£ mums magma as» msoaumuosmw so>mm 0cm soon you wdasouw Houm¢

 

umo:0¢« o£u mo Hm>osuu comm mua>auom umwuso mo uaosousmmmz

NHN mgm<8

154

decrease in the level of all three enzymes indicated that
the mutant was not constitutive for them.

Effect of Protein Inhibitors on the Utilization of
§;Mannose and L-Rhamnose- To show further that the first
three enzymes were not constitutive, the inducers L-mannose
and L-rhamnose were added back to the mutant cells prev-
iously grown in the absence of inducers and the rate of
their utilization measured manometrically. To distin-
guish between constitutive and newly induced enzymes,
either chloramphenicol or puromycin was added to the
reaction. As Figures 47 and 48 indicate, in the absence
of these inhibitors L-rhamnose was readily utilized and
reached a maximal rate comparable to D-glucose. L-
Mannose also was oxidized but at a slower rate than L-
rhamnose. In the presence of either chloramphenicol or
puromycin neither Irmannose nor L-rhamnose was utilized.
The results thus showed that the oxidation of L-mannose
and L-rhamnose by the mutant cells was due to the bio-
synthesis of new enzymes and not to a constitutive muta-
tion.

The results indicated that another mechanism was
Operating within the cell to regulate growth on Lsmannose.
Since dihydroxyacetone phosphate was readily metabolized,

L-glyceraldehyde was restudied to determine if it exerted

 

155

Fig. 47. Effect of chloramphenicol and puromycin

on enzyme induction and hexose utilization in the
mutant after four generations of growth in the

absence of inducer. Each.Warburg vessel contained

in a volume of 0.55 ml, 10 pmoles of either L-rhamnose
or L-mannose, 180 pmoles of KOH (center well), 1000

pg of chloramphenicol or 500 pg of puromycin, and
about 2 mg dry weight of cells. As a control water
was substituted for the inhibiter of protein biosyn-
thesis and for endogenous activity water was sub-

stituted for the hexose.

Figure 47 .

 

 

  
 
  
    

 

 

 

 

 

0 "7'"
\ 5 D-Glucose
3 (.3 & ° . L-Bhamnose
<3 o—l <——/
E! m
g o
A 2 . -
3'; 5 0— ~ ‘ \K L Mannose
. 2 >4
a) E .
f! V N— Endogenous
H .
1 g) ' / . I ‘ I
0 4o 80 120
MINUTES
50"“ CHLOBAMPHENICOL
\
“:3
g o
A L-Rhamnose
SS 25 .
a, N L-Manno se
is a:
3 a \ Endogenous
H
"=13 I I
0 40 80 120
MINUTES
50 .—
\m . PUBOMYCIN
8E}:
E—c 0
Egg N D-Glucose
2 ~—— -Rh
0 5 L amnose \
>4
'0 m
s c:
3V ‘ L M
- annose
: g) \ Errdogenous
:1 , I
0 “0 .«'80 120

MINUTES
'156’

157

Fig. 48. Effect of chloramphenicol and puromycin on
enzyme induction and hexose utilization in the mutant
after seven generations of growth in the absence of

the inducer. Refer to Figure 47 for details.

Figure 48.

uliters 02 UPTAKE/

mg (DRY WT) CELLS

UPTAKE/
mg (DRY WT) CELLS

uliters O

uliters O UPTAKE/

mg (DRY Hf),CELLs

50

25

50

25

 

 

    
     
  

__
°D-Glucose ' L-Bhamnose
.‘K/ L-Mannose
\u
- . v~—Endogenous
0 40 80 120
MINUTES
7. CHLOBAMPHENICOL
f’,,D-Gluoose
T—

L-Rhamnose..:§

L-Mannose
“~—Endfigenous

 

 

0 40 80 120

50

25

MINUTES

PUROMYCIN

D-Gluoose
<"’/

L-Rhamnose

\

L-Mannose
K- Endogenoui
1

     
  
  

 

O 40 80 120

MINUTES
158

159

any effect on the cells. Part I of this thesis showed
that L-glyceraldehyde underwent oxidation to glyceric
acid and reduction to glycerol. Thus, investigations
were conducted to establish if these reactions were
instrumental in regulating Limannose metabolism. Since
the wild-type cells completely utilized L-mannose with—
out growth, the possibility existed that a degradative
product of L—mannose which inhibited growth accumulated
in the growth medium. Attempts to find such a product
failed.

Contribution of Glyceric Acid to L-Glyceraldehyde

Metabolism- The enzyme which converts L-glyceraldehyde

 

to glyceric acid was detected only by identification of
the reaction product (see Part 1). Paper Chromatography
of the growth media obtained.from both wild-type and
mutant cells actively utilizing L-mannose showed the
accumulation of a compound which co-chromatographed with
DL-glyceric acid. NWhether or not some of the glyceric
acid was metabolized by these cells is not known.. How—
ever, since it did accumulate as an excretory product of
Irmannose degradation by both cell types, it was eliminated
as a possible growth inhibitor.

Effect of Glycerol on the Growth of the‘Wild—Type

and Mutant Cells- Both the mutant and the wild-type cells

160

grew equally well on glycerol (Figure 49). Although the
reduction of L-glyceraldehyde to glycerol could not be
assayed in crude extracts because of interfering reac-
tions, partial fractionation with ammonium sulfate yielded
a 45-60% fraction.which showed reduction Of L-glyceralde-
hyde. This enzyme was detected in fractionated extracts
Obtained from both wild-type and mutant cells previously
grown on L-rhamnose or Lsmannose respectively (Table XX).
In addition, L-mannose also induced the enzyme in wild-
type cells after they were previously grown on D-glucose.
Since both L—hexoses induced the reductase in both cell
types, the reduction of Lpglyceraldehyde to glycerol was
not considered to be an influential reaction in inhibiting
the growth Of the parent strain on Lemannose.

Effect of DL-Glyceraldehyde on the Growth of the
Wild-Eype and Mutant Cellg: DL-Glyceraldehyde inhibited
the growth of both wild-type and mutant cells above a
concentration of 0.007 mM. Figure 50 compares the effect
of DL-glyceraldehyde on the growth of the cells. In
neither case was the inhibitory effect overcome at the
higher concentration. No conclusion4was drawn as to
which enantiomorph caused the growth inhibition.

grgwth of the Wild gype and Mutant on L—Galactose-

E, aerogenes re5ponded to growth on L-galactose, a rare

 

161

Fig. 49. Growth Of E, aerogenes on glycerol.

Figure 49.

 

 

0.8 __ 0. Wild Type
O-Mutant
0.6.—
O
l-r
E“
0.
O
0.4_.
0.2_
l_ I l l l l
O 2 4 6 8 10 12
HOURS

162

 

163

TABLE XX
lnduction of L-giyperaldehyde reductase
Reductase activity was measured using the routine assay. The

enzyme was from a 45-60% ammonium sulfate fraction.

 

 

 

Cell Type Growth Substrate Reductase Activity*
Wild L-Rhamnose 0.088
D-Glucose and
IrMannose 0.020
D-Glucose 0.000
Mutant IwMannose 0.021
D-Glucose 0.000

 

* Activity is expressed as pmoles NADH oxidized/min/mg of protein.

164

Fig. 50. Growth Of E, aerogenes in nutrient broth
supplemented with varying amounts of DL—glyceralde-

hyde.

Figure 50.

 

0H 0H

0

2s 4H0.0

28 500.0:l/K

    
   

820902

mmDOm

N.o

:.0

0.0

 

 

as 0H0.0_\\\»

mmwa QAHz

   

N.0

0.0

 

owS.q.O

165

166

hexose, in the same way as it did to L-mannose. Although
L-galactose and L-mannose differ structurally, they both
undergo similar biodegradative patterns and produce L-
glyceraldehyde as a major intermediate. L-Galactose

metabolism has been established in.E, aerogenes (Mayo and

 

Anderson, unpublished results) and found to be isomerized
to L-tagatose, phOSphorylated with ATP to L-tagatose-l-P,
and cleaved to dihydroxyacetone phosphate and L-glyceral-
dehyde. If the basis for growth on Limannose involved
L—glyceraldehyde or a subsequent metabolic intermediate,
then the L-mannose positive mutant should also grow on
L-galactose more readily than the wild type. As Figure
51 indicates, a 754hour lag preceded growth of the mutant
on L-galactose whereas the wild type showed no appreciable
growth even after 120 hours. There was some initial
growth by both cell types but this was attributed to an
unknown contaminent rather than to growth on L-galactose.
The mutant cells after growing on L-galactose also grew
on L-mannose.
DISCUSS ION

The growth of E, aerogenes on Lamannose involves a
mechanism other than a constitutive mutation for the,
first three enzymes in the pathway. The evidence in this

investigation suggests that the basis for the gain in the

[Eu 4-

167

Fig. 51. Growth of E. aerogenes on L-galactose.

Figure 51.

 

 

 

 

00000
04H 40 0: 0 00H
IILNoO
110.0
110.0
1
924902 _
L moo

mama and:

 

30°

0.0

 

.moO

0175.0.0 '

168

169

ability of the mutant to grow on Lsmannose lies in its
ability to metabolize L-glyceraldehyde or a subsequent
metabolic intermediate. The growth of both the wild type
and the mutant was inhibited when the cells began to uti-
lize Lemannose, but only the mutant overcame the inhibi-
tion. This suggested that a compound accumulated within
the cells which inhibited their growth rather than the
selection of a mutant strain which resisted the inhibi-
tory effect Of this product. Since Lemannose induced the
isomerase, kinase, and aldolase in both types Of cells,
the first three enzymes in the biodegradative pathway
were not influential in regulating growth of the cells
on Lemannose. In addition, none Of the enzymes was
constitutive when the inducer was removed.
L-Glyceraldehyde inhibits a variety of cellular
processes but the exact site(s) Of inhibition are still
questionable. .Mendel (74) was the first to report that
L-glyceraldehyde inhibited the formation of lactic acid
from glucose in tumor cells. Rudney (75) had shown that
hexokinase in rat skeletal muscle, rat sarcoma, beef brain,
and yeast also was inhibited by L-glyceraldehyde. In a
later report Wenzel, Joel, and Oelkers (76) demonstrated
that Lpglyceraldehyde inhibited glycolysis in Ehrlich

ascite tumor cells. On the other hand, D-glyceraldehyde

170

has been implicated by Rapkine g£_gl_(77) to inhibit D-
glyceraldehyde-3-P dehydrogenase. Possibly DL-glyceral-
dehyde is working in a dual capacity to inhibit growth

of E, aerogenes. Lardy and coworkers (78) have shown that
rabbit muscle aldolase condenses L-glyceraldehyde and
dihydroxyacetone phosphate to yield L—sorbose-l-P which
strongly inhibits hexokinase. Whether or not a similar
reaction occurs in bacteria is not known. Possibly the
bacterial glycolytic aldolase is capable of condensing

the products Of the L—fructose-l-P cleavage producing L-
sorbose-l-P which may be toxic to the cell. If this is
the case, the mutant may overcome this toxicity by synthe-
sizing an enzyme which converts storbose-l-P to a common
metabolite.

Although L-glyceraldehyde is simultaneously reduced
to glycerol and oxidized to glyceric acid, the enzymes
were present in both wild type and mutant. Both cell
types grew readily on glycerol and when grown on L-
mannose, glyceric acid accumulated in the growth medium.
Thus, these reactions were not considered to be significant
in regulating Lemannose metabolism.) The mutant cells,
however, were capable of growing on L-galactose more

readily than the wild type and Lnglyceraldehyde is a

 

171

common product Of both L-hexoses.

Previous investigators have shown that the meta-
bolism of unnatural sugars involved a constitutive mutation
not for the synthesis Of new enzymes but rather for the
production of a non-specific enzyme capable of converting
the unnatural substrate to a common metabolic intermediate.
Mortlock §£_§E_(7) have shown that growth of E, aerogenes
on xylitol and L-arabitol was explainable by the selection
Of a mutant constitutive for ribitol dehydrogenase, which
converted xylitol and Learabitol to D- and L-xylulose,
resPectively. Similarly, growth on L-xylose was due to
selection Of a mutant constitutive for L-fucose isomerase
(2). In contrast, D-lyxose appeared to be an exception
to this generalized phenomenon. Allison and Anderson (4)
reported that D-lyxose was isomerized to D-xylulose but
they were unable to isolate constitutive mutants for D-
lyxose isomerase. The enzyme was induced only by D-lyxose,
and although D-mannose also was isomerized by this same
enzyme, it was unable to induce it. Lin gt §E_(6)
reported that two genetic events regulate the metabolism
of mannitol in E, aerogenes. The suppression of the
phosphotransferase pathway by which mannitol was normally
metabolized gave rise to a constitutive mutation for the

production of D-arabitol dehydrogenase which converted

172

mannitol to D-fructose. Other systems involving the
utilization of an unnatural substrate by derepression of
an enzyme include: altrose-galactoside via a-galactosi-
dase (79), B-glycerolphosphate via alkaline phOSphatase
(80), and putrescine via diamine41-keoglutarate transami-
nase (81). The enzymes of L-mannose metabolism also are
non—Specific in that they degrade the naturally occurring
hexose, L-rhamnose. However, unlike the previously men—
tioned cases, the first three enzymes involved in L-
mannose metabolism are not constitutive.

To conclude, both mutant and wild-type cells utilized
L-mannose and their growth was inhibited by a product of
L-fructose-l-P. This inhibitor may be L-glyceraldehyde
or some product thereof. However, only the mutant was
able to overcome the inhibition resulting in active growth
on L-mannose.

SUMMARY

The biodegradative pathway of L-mannose by E, aerogenes
PRL-R3 has been elucidated. LeMannose is isomerized
to L-fructose which is phosphorylated with.ATT and a
kinase to L-fructose-l-P. LPFructose-l-P is cleaved
by an aldolase to dihydroxyacetone phosphate and L- .

glyceraldehyde. L-Fructose and a new sugar phOSphate,

173

L-fructose-l-P, were isolated and identified, and an
alternative procedure was develOped for the preparation
of L-fructose. The enzymes in the pathway have been
characterized. They were induced by either Lemannose

or L-rhamnose and the biodegradation of these two
L-hexoses has been shown to involve the same enzymes.

A mutant strain of E, aerogenes has been isolated,

which, unlike the wild type, grows readily on Lemannose

as the sole carbon source. The mutant is not constitutive
for the firstthree enzymes in the biodegradative pathway.
Thus, the basis for growth of the mutant on Lemannose
possibly lies in its ability to metabolize L-glyceralde-

hyde or a product thereof.

10.

11.

l2.

13.

14.

15.

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RES

4441)) 444444)”