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THE ROLE OF PY RUVATE KINASE IN THE REGULATION

OF GLYCOLYS I S AND GLUCONEOGENES IS

I N PROPION I BACTERIUM SHERMANI I

A thes i s presented i n parti al ful fi l ment of

the requi rements for the degree of Doctor of

Phi l osophy i n B iochemi stry at Mas sey

Uni vers i ty, New Zeal and .

John Beresford SMART

1980

ii .

ABSTRACT

Pyruvate kinase catalyses the main irreversibl e reaction of glycol ysis

in Propionbacterium shermanii since the ATP-dependent phosphofructokinase

is l argel y repl aced by a pyrophosphate-dependent phosphofructokinase

catalysing a reversibl e reaction . Measurement of activity of several

glycolytic enzymes in gl ucose- , gl ycerol - and l actate-grown cel ls showed

that pyruvate kinase activity is much higher than that of pyruvate ,

orthophosphate dikinase which catalyses the reversible interconversion

of PEP and pyruvate .

This poses probl ems in cel l s grown on l actate where gl uconeogenesis

must operate to supply hexoses and pentoses for biosynthesis . The

regul atory properties of this enzyme were accordingly studied .

The partial ly purified ( 140-fol d ) pyruvate kinase displ ayed sigmoidal

kinetics for both of its substrates , phosphoenol pyruvate { PEP) and ADP .

At pH 7 . 5 the interaction coefficient ( nH ) for PEP saturation in the

absence of effectors was in the range 1 . 9-2 .5 \'thil e for ADP saturation it

was 1 . 7-2 . 1 . The pyruvate kinase was shown to be activated by gl ucose

6-phosphate (G6�at non-saturating ( 0 . 5 mM) PEP concentrations but other

glycolytic and hexose monophosphate pathway intermediates and AMP were

without effect. Hal f-maximal activation was obtained at 1 mM G6P . The

presence of G6P decreased both the PEPo . 5v and ADPo . sv val ues and the

Hil l interaction coefficient for each substrate . The enzyme was strongly

inhibited by ATP and inorganic phosphate ( Pi ) at a l l PEP concentrations .

At non-saturating ( 0 . 5 mM) PEP hal f-maximal inhibition was obtained at

1 . 8 mM ATP and 1 • 4 mM Pi . The inhibition of both Pi and ATP cou ld be

l argely overcome by G6P . The G6P activation and other regul atory

properties of the enzyme were pH dependent .

i i i .

On the bas i s of th i s i n v i tro study i t was suggested that the

acti vi ty of pyruvate k inase i n v i vo i s determi ned by the bal ance

between activators and i nh i bi tors such that i t i s i nh i bi ted by ATP

and Pi during gl uconeogenes i s whi l e , duri ng gl ycolysi s , the i nh i bi tion

i s rel i eved by G6P . Such a mechani sm requi res that the G6P concentration

shoul d be sufficientl y high when cul tures are grown on gl ucose or glycerol

but not on l actate .

To veri fy thi s proposed mechani sm the i n vi vo concentrati ons of

a number of sel ected g lycolyti c i ntermedi ates were measured in P . sherman i i

growi ng under a range of nutri ti onal condi ti ons usi ng batch and

conti nuous cul tures . The pyruvate ki nase acti vator , G6P , was mai ntai ned

at l evel s of 1-2 mM i n l actate-grown cel l s but , when growi ng i n the

presence of h i gh l evel s of g l ucose or glycerol , G6P was present at l evel s

between 5 and 16 mM i n the cel l .

F6P and FBP were a lways present at l evel s bel ow 1.0 mM and 0.2 mM

respecti vely i n l actate- and gl ucose-grown ce l l s but attai ned s i gni fi cantly

h igher l evel s on gl ycerol -grown cel l s . At hi gh concentrations of

gl ucose where h i gh G6P l evel s were found , F6P was present at concentrati ons

much l ower than woul d be expected from the equi l i br i um constant of the

phosphogl ucoi somerase ; thi s was not the case when h i gh G6P l evel s were

attained by growth on glycero l . The reason for thi s i s not known .

level s of the i nh i b i tor of pyruvate k i nase acti v ity , ATP , were mai ntai ned

i n the range 1-2 nf.t under al l the condi ti ons studi ed.. At the in vi vo

l evel s of the substrates PEP and ADP thi s l evel of ATP woul d not by i tsel f

mai nta i n pyruvate k i nase i n an i nacti ve state i n the presence of the

1 -2 mM G6P shown to be present i n l actate-grown cel l s . The other inhi bi tor,

Pi , must al so be important but the _!n vi vo l evel s of thi s effector were

not determi ned .

iv.

A reinvestigation of the pyruvate kinase at the in vivo level s of

substrates ( PEP and ADP ) and effectors (G6P and ATP) found in the ce l l

indicated that . at l evel s above 10 mM Pi• concentrations of 5-15 mM G6P

( as found in cel ls growing on gl ucose and glycerol ) were required to

activate the enzyme whil e concentrations of 1-2 mM G6P ( as found in

l actate-grown cel ls ) were not ab l e to overcome the inhibition by ATP and Pi •

A concentration of 10 mM P ; in the cel l does not seem unreasonabl e and

at this concentration the proposed mechanism for control of the pyruvate

kinase �vivo woul d be abl e to operate .

During this investigation data were also col l ected on growth yiel ds

and carbon bal ances in both batch and continuous cul tures during growth

on the three substrates l a ctate . gl ucose and gl ycerol .

Succinate was found to account for up to 26% of the products in

gl ucose cul tures but was only present at negligibl e l evels in l actate

cul tures. Accumul ation of succinate in the gl ucose cul tures was

highest in the l ater stages of growth of batch cul tures and in carbon­

limited continuous cul tures when the G6P l evel was l ow. This suggests

that regul ation of pyruvate kinase by G6P may a lso serve to determine

the rel ative fl ux via the PEP : carboxytransphosphoryl ase and pyruvate

kinase enzymes - the carboxytransphosphoryl ase functioning to provide

PP; for the PP; -dependent phosphofructokinase in glycolysis .

I t is a lso proposed that G6P may be invol ved in partitioning g l ucose

metabolism via g lycolysis and the hexose monophosphate pathway - high

internal G6P concentrations favouring fl ow through the hexose monophosphate

pathway and thus bypassing the PP; -dependent phosphofructokinase - however .

this is highly specul ative .

ACKNOWLEDGEMENTS

I wish to thank my Supervisor , Dr G . G . Pri tchard , for h is

i nval uabl e advi ce , encouragement and assi stance throughout the

course of thi s study.

I woul d also l i ke to thank Or J .C . Hawke and Or I . G. Andrew

for thei r techn ical advi ce on the gas-l i qu i d chromatographi c

analyses ; Or J. Thompson o f the Dai ry Research I nsti tute ,

Pa lmerston North , for h is advi ce concerning the fl uorometri c

analyses of metabol i tes and the Appl i ed B i ochemi stry Di vi s ion of

v .

the D.S . I.R. , Pa lmerston North , for the use o f thei r SPF 500 Spectra­

fl uorometer.

In addi ti on , parti cul ar thanks are extended to Mrs Gl enda Shaw

for typi ng the manuscri pt and to Mrs Cl a i re Smart for her unfai l i ng

support and assistance throughout the i nvesti gati on and in the

preparation of this thesis .

L I ST OF CONTENT S

Abstract

Acknowl edgements

l i st of Contents

li st of Fi gures

l i st of Tab les

li st of Abbrevi ations

CHAPTER 1 - GENERAL I NTRODUCTION

1 . 1

1 .2

1 . 3

Regul ati on of carbohydrate metabol i sm i n bacteri a

1 . 1 . 1 Rol e of pyruvate k i nase i n regul ati ng

carbohydrate metabol i sm i n bacteri a

Carbohydrate metabol i sm i n propion i c aci d bacteri a

Aims of th i s i nvesti gati on

CHAPTER 2 - MATERIAL S AND METHODS

2 . 1

2 . 2

2 . 3

2 . 4

2 . 5

2 .6

2 . 7

Introduct ion

Materi al s

Organi sm and mai ntenance

Determination of the rel ati onsh i p between opti cal

dens i ty and dry wei ght of cel l suspens ions

Estimati on of substrates and products i n the growth

med i um

2 . 5 . 1 Glycerol

2 . 5 . 2 lactate and succi nate

2 . 5 . 3 Acetate and propi onate

2 . 5 . 4 Gl ucose

Protei n esti mation

Measurement of enzyme acti vi ti es

Page Number i i

V

vi

xi i

x iv

xvi

1

3

1 1

2 1

24

24

25

26

28

28

28

29

29

30

30

2 .8

2 . 9

Determination of glycolyti c i ntermedi ates by

enzymi c/fl uorometri c techni ques

Estimati on of cel l water vol ume

CHAPTER 3 - GROWTH CHARACTERI STICS OF PROPION IBACTER IUM

SHERMANI I ON THREE DI FFERENT CARBON SOURCES

3 . 1

3 . 2

3 . 3

3 . 4

3 . 5

3 . 6

I ntroducti on

Composi tion of the defi ned medi um

Growth condi ti ons

Growth of P . shermani i on the defi ned medi um

Enzyme l evel s i n batch cul tures of P . shermani i

Di scu ssion

CHAPTER 4 - PYRUVATE KI NASE

4 . 1

4 . 2

4 . 3

I ntroduction

4 . 1 . 1

4 . 1 . 2

4 . 1 . 3

Mammal i an pyruvate ki nases

Bacteri al pyruvate k i nases

P . shermani i pyruvate k i nase

Pyruvate k i nase puri fi cati on

4 . 2 . 1

4 . 2 . 2

4 . 2 . 3

4 . 2 .4

4 . 2 . 5

4 . . 2 .6

4 . 2 . 7

Growth and harvest o f Propioni bacteri um

shermani i

Breakage of cel l s and preparati on of

cel l -free extract

Streptomyci n sul phate treatment

Ammon i um sul phate preci pi tation

DEAE - Sephadex i on exchange chromatography

Gel fi l trati on on Sephacryl 5200

D i scuss ion of the puri fi cati on scheme

Properties of the parti al ly puri fi ed pyruvate k inase

from P. s hennani i

4 . 3 . 1 Pyruvate-ki nase assay

vi i .

33

36

39

39

40

41

45

47

49

49

50

54

54

54

55

57

57

58

58

61

65

65

4. 3 . 2

4. 3 . 3

4. 3 . 4

4. 3 . 5

4. 3 . 6

4. 3 . 7

4. 3 . 8

4. 3 . 9

4. 3 . 10

4. 3 . 11

4. 3 . 12

4. 3 . 13

4. 3 . 1 4

Treatment o f k i neti c data

Effect of enzyme concentration on acti vi ty

Stud ies on the stabi l i ty of pyruvate ki nase

pH profi l e of pyruvate ki nase

Monoval ent cati on requi rement of pyruvate ki nase

Effectors of pyruvate ki nase acti vi ty

Rel ationshi p between pyruvate ki nase acti vi ty

and varyi ng PEP concentrat i on

4. 3 . 8 . 1

4. 3 . 8 . 2

E ffect o f ADP concentration o n the

PEP saturation curve

Effect of 2 .0 mM G6P on the PEP

saturation.curve

Rel ationsh i p between pyruvate ki nase acti vi ty

and varyi ng ADP concentrati on

4. 3 . 9 . 1

4. 3 . 9 . 2

Effect of PEP concentrati on on the

ADP saturation curve

Effect of 2 . 0 mM G6P on the ADP saturation curve

Effect of GDP on pyruvate k i nase acti v i ty

Effect of Mg++ on pyruvate ki nase acti v i ty

vi i i .

66

68

70

70

72

74

80

80

83

84

84

87

87

88

4. 3 . 11 . 1 Effect of Mg++ on pyruvate ki nase 90

acti vi ty of P . shermani i

4. 3 . 11 . 2 Effect of G6P on Mg++ acti vat ion 90

of pyruvate kinase

4. 3 . 11 . 3 I nteraction between Mg++ and ADP 92

Effect of Mn++ on the PEP saturation curve

of pyruvate k i nase

Effect of varyi ng G6P concentration on

pyruvate ki nase activi ty

Effect of varyi ng ATP concentration on

pyruvate k i nase acti vi ty

94

96

96

4.3 . 15

4. 3 . 16

4. 3 . 17

4 . 3 . 18

4. 4 Di scuss ion

Effect of varyi ng P; concentration on

pyruvate k i nase activ i ty

Interacti on between G6P and ATP or P; i n the rel ati ons hi p between pyruvate k i nase

acti vi ty and PEP concentration

Effect of pH on the ki neti c and al l oster ic

properties of pyruvate ki nase

i x .

100

100

101

4 . 3 . 17 . 1 Effect of pH on the PEP saturation 104

c urve

4 . 3 . 1 7 . 2 Effect of G6P and ATP on the PEP 106 saturation curve at pH 6 . 0

4. 3 . 17 . 3 Rel ati ons hi p between pyruvate 108

ki nase acti vi ty and ADP concentration

at pH 6 . 0

4 . 3 . 17 . 4 Effect of G6P on the pH profi l e 108 of pyruvate ki nase

Some kineti c data on the pyruvate ki nase i n

a crude extract from P . s hermani i cel l s

1 10

112

CHAPTER 5 - Jli V IVO LEVELS OF METABOL ITES IN P . SHERMAN I I GROWI NG

UNDER D I FFERENT NUTRITIONAL CONDIT IONS

5 . 1

5 . 2

5 . 3

I ntroducti on

Compari son of metabol i te l evel s in resti ng cel l

suspens i ons us i ng 14c-l abel l ed carbon sources

5 . 2 . 1

5 . 2 . 2

Preparation of resti ng cel l s uspensions

Label l i ng and separation of glycolyti c

i ntermedi ates

�vi vo concentrati ons of metabol i tes i n batch and

conti nuous cul tures of P . shermani i

5 . 3 . 1

5 . 3 . 2

Compari son o f extraction procedures for

analysi s of glycolyti c i ntermedi ates

Batch cul ture stud ies

1 18

1 18

1 20

121

125

127

1 30

5 . 3 . 2 . 1 Prel imi nary i nvesti gati on of i n vi vo 130

concentrati ons of metabol i tes

5 . 3 . 3

5 . 4 D i scussion

5 . 3 . 2 . 2 Detai l ed study of metabol i te l evel s on batch c ul tures of

P . shermani i

Conti nuous cul tures

5 . 3 . 3 . 1

5 . 3 . 3 . 2

5 . 3 . 3 . 3

Carbon bal ances duri ng conti nuous

growth on l actate , gl ucose and

glycerol defi ned med ia

I n v ivo concentrati ons of meta­

bol i tes i n conti n uous cul tures

Spec if ic acti v i t i es of sel ected

enzymes i n cont i nuous cul tures

CHAPTER 6 - THE ROLE OF G6P I N THE REGULATION OF METABOL I SM

OF P . SHERMAN I I

6 . 1

6 . 2

6 . 3

6 . 4

I ntroducti on

Rel ationshi ps between external carbon source

concentrati on and G6P or pyruvate l evel s

6 . 2 . 1

6 . 2 . 2

6 . 2 . 3

Rel ati onshi p between external gl ucose

concentrati on and i nternal G6P concentration

Rel ationshi p between external glycerol

concentrati on and i nternal G6P concentration

Rel ati ons hi p between external l actate

concentration and i nternal pyruvate

concentrati on

Rei nvesti gati on of the regul ati on of pyruvate k i nase

acti vi ty at i n vi vo concentrati ons of s ubstra tes and effectors

6 . 3 . 1

6 . 3 . 2

Di scussion

Parti al puri fi cati on of pyruvate k i nase

Studi es on the parti a l ly puri fi ed pyruvate

ki nase

x .

131

137

137

141

144

147

153

153

153

154

158

160

160

162

165

CHAPTER 7 - SOME ADDITIONAL ASPECTS OF METABOL I C REGULATION I N P .SHERMANI I

7 . 1

7 . 2

7.3

7.4

Introduction

The effect of G6P concentration on G6 P dehydrogenase

acti v i ty

The effect of carbon source on the uptake of phosphate

Growth and metabol i sm of P . shermani i on a mi xed gl ucose/

l actate defi ned med ium

CHAPTER 8 - GENERAL DI SCU SSION

8 . 1

8 . 2

8.3

Regul ati on of pyruvate k i nase duri ng glycolys i s and

g l uconeogenesi s

Further pos si bl e rol es of G6P as a regul ator of carbohydrate metabol i sm in P.shermani i

Areas for further study

Bi bl i ography

xi .

167

167

170

174

178

184

19 1

196

L I ST OF F IGURES

Fi gure Number Ti tl e

1 . 2 Carbohydrate metabol i sm i n P . shermani i .

2 . 4 Rel ationshi p between dry wei ght and opti ca l

density.

2 . 8

3 . 4

4 . 2 . 1

4 . 2 . 5

4 . 2 . 6

4 . 3 . 2

4 . 3 . 3

4 . 3 .4

4 . 3 . 5

4 . 3 . 7

4 . 3 .8

4 . 3 . 9

4 . 3 . 10

4 . 3 . 11

4 . 3 . 1 1 . 3

4 . 3 . 12

Fl uorometri c analys i s of metabol i tes .

Growth of P . sherman i i i n the defi ned medi um

on lactate , gl ucose and glycerol .

Speci fi c acti vi ty of pyruvate ki nase duri ng

growth of P . shermani i .

DEAE - Sephadex i on exchange chromatography.

Sephacryl S200 ge l fi l tration .

Treatment of ki neti c data .

Effect of enzyme concentrati on on acti vi ty .

Stabi l i ty of pyruvate k inase .

pH profi l e of pyruvate ki nase .

Effect of acetyl phosphate concentration on

pyruvate ki nase acti vi ty.

Effect of ADP on the rel ati onsh i p between

acti vi ty and PEP concentrati on .

Effect of PEP on the rel ati onsh i p between

acti vi ty and ADP concentration.

Effect of GDP concentrati on on pyruvate ki nase

acti vi ty.

Effect of Mg++ concentrati on on pyruvate ki nase ' acti vi ty.

Effect of t1g++ concentrati on on the rel ati onsh i p

between pyruvate ki nase acti vi ty and ADP

concentration .

Effect of MnCl 2 concentration on the PEP satur­

ation curve .

Page N umber

13

27

35

42

56

59

60

67

69

71

73

79

81

85

89

91

93

95

4 . 3. 13

4 . 3 . 14

4 . 3 . 15

4 . 3 . 16

4 . 3 . 17 .4

5 . 2 . 2

5 . 3 . 2 . 2

6 . 2

6 . 2 . 3

6 . 3 . 2

7 . 2

7 . 4

xi i i .

Effect of G6P concentrati on on pyruvate

ki nase act iv i ty.

Effect of ATP concentrati on on pyruvate

ki nase acti vi ty.

Effect of Pi concentrati on on pyruvate

k i nase acti vi ty.

Interaction between G6P and ATP or Pi on the

PEP saturation curve .

Effect of G6P on the pH profi l e of pyruvate

k i nase .

Autoradi ograph of 14c-l abel l ed i ntermedi ates .

Growth of P . shermani i i n batch cul tures .

Rel ationsh i p between i nternal G6P concentration

and external gl ucose or gl ycerol concentration .

Rel ationshi p between i nternal pyruvate

concentrati on and external l actate '

concentrati on.

Pyruvate ki nase acti vi ty at in v i vo concentrati ons of substrates and effectors .

Rel ationshi p between gl ucose 6-phosphate

dehydrogenase act iv i ty and G6P concentrati on .

Growth of P . s hermani i o n mi xed l actate/gl ucose

medi um.

97

98

98

102

109

123

134

157

157

163

169

176

Tabl e Number

3 . 4

3 . 5

4 . 1 . 2

4 . 2

4 . 3 . 7

4 . 3 . 8

4 . 3 . 9

4 . 3 . 14

4 . 3 . 16

4 . 3 . 1 7 . 1

4 . 3 . 1 7 . 2

4 . 3 . 18

5 . 3 . 1

5 . 3 . 2 . 1

LIST OF TABLES

Ti tl e Page Number

In text .

Enzyme l evel s i n batch cul tures of

P . shermani i .

Summary of k i neti c properti es of some

bacteri al pyruvate ki nases .

Puri fi cation of pyruvate ki nase from 40 g wet

packed wei ght of P . shermani i cel l s .

Effect of metabol i tes on pyruvate ki nase

acti v i ty.

Effect of ADP on the rel ati onshi p between

acti vity and PEP concentrati on .

Effect of PEP on the rel at ionshi p between

acti vi ty and ADP concentrati on .

Effect of ATP concentrati on on pyruvate

ki nase acti vi ty.

The effects of G6P , ATP and Pi on the

rel ationshi p between pyruvate ki nase acti vi ty

PEP concentrati on .

The effect of pH on the rel ati onshi p between

pyruvate k i nase acti vi ty and PEP concentrati on .

The effect of G6P and ATP on the rel ationshi p

between pyruvate k i nase acti vi ty and PEP concentration at pH 6 .0 .

Ki neti c data on pyruvate ki nase i n a crude

extract of P . shenmani i .

Effect of extracti on procedures on i n v i vo

metabol i te l eve l s .

I n vi vo concentrati on of metabol i tes i n batch

cul tures .

43

46

53

62

75

82

86

99

103

105

107

111

129

132

5 . 3 . 2 . 2

5 . 3 . 3 . 1

5 . 3 . 3 . 2

5 . 3 . 3 . 3

5 . 4

6 . 2 . 1

6 . 2 . 2

6 . 2 . 3

6 . 3 . 1

6 . 3 . 2

7 . 3

7 . 4

Batch cul tures of P . shermani i on l actate , gl ucose and gl ycerol defi ned medi a .

Carbon bal ances i n P . s herman i i conti nuous

cul tures .

I n v i vo l evel s of metabol i tes i n continuous

cul tures .

xv.

135

1 39

142

Enzyme l eve l s i n continuous cul tures . 146

Summary of data on i n vi vo l evel s of metabol i tes 148

i n P . shermani i .

Rel ati onshi p between external gl ucose concen- 155

trati on and i nternal G6P concentration .

Rel ations hi p between external g lycerol concen- 156

trati on and i n ternal G6P and F6P concentrations .

Rel ati onshi p between external l actate concentr- 159

ati on and i nternal pyruvate concentrat ion .

Partia l puri fi cation of pyruvate k i nase . 161

Effect of i norgani c phosphate on the cooperati vi ty 164 of G6P acti vati on of pyruvate k i nase .

Effect of carbon source o n phosphate uptake i n P . shennani i .

Growth of P . s hermani i on mi xed gl ucose/l actate defi ned medi um.

173

175

ADP

AMP

ATP

Bi s tri spropane

COP

CM-

CoA

DE- , DEAE-

OHAP

DNase

EO

EDTA

EMP

FBP

F6P

Ga 3-P ,

Glyceral dehyde 3-P

GDP

g lycerol 3-P

G6P

GTP

HE PES

HMP

lOP

MES

NAD

NADH

L IST OF ABBREVIATIONS

adenos i ne 5 ' -d i phosphate

adenosi ne 5 ' -monophosphate

adeno s i ne 5 ' -tri phosphate

2 b i s/-tri s ( hydroxymethyl ) methyl ami no_]-propane

cyti di ne 5 ' -di phosphate

carboxymethyl -

coenz}111e A

d i ethyl ami noethyl -

di hydroxyacetone phosphate

deoxyri bonucl ease

Entner-Doudoroff

ethyl ened i ami ne tetra-aceti c aci d

Embden -Meyerhof-Parnas

fructose 1 ,6-bi sphosphate

fructose 6-phosphate

Jglyceral dehyde 3-phosphate ) guanos i ne 5 ' -d i phosphate

glycerol 3-phosphate , a-gl ycerophosphate

gl ucose 6-phosphate

guanos i ne 5 ' -triphosphate

N-2-hydroxyethyl pi peraz i ne-N ' -2-ethanesul phon i c a ci d

hexose monophosphate

i nosi ne 5 ' -d i phosphate

2�N-morphol i no_lethane sul phoni c aci d

ni coti nami de aden i ne d inucl eotide

ni coti namide aden i ne d i nucl eoti de , reduced

NADP

NADPH

nH 0°540

OAA

PEP

6-PG

3-PGA · pi

PP;

ri bose 5-P

ri bul ose 5-P

Trici ne

Tri s

UDP

Ymax

E. coli

N . crassa

S . l acth

xvi i .

n i coti nami de adeni ne di nucl eotide phosphate

n i coti nami de adeni ne di nucl eotide phosphate , reduced

Hi l l interaction coeffi ci ent

opti cal dens i ty at 540 nm

oxal oacet i c aci d

phosphoenol pyruvate

6 -phosphogl uconate

3-phosphoglyceri c aci d

i norgan i c phosphate

i norgani c pyrophosphate

ri bose 5-phosphate

ri bul ose 5-pho sphate

N-tri s ( hydroxymethyl } methyl glyci ne

tri s ( hydroxymethyl } ami nomethane

uri di ne 5 1 -di phosphate

maximum veloci ty

Escheri ch i a col i

Neurospora cras sa

Streptococcus l a cti s