the role of pyruvate kinase in the regulation of
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Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.
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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
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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 .
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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 .
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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 .
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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 .
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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