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I N PROPION I BACTERIUM SHERMAN I I
A thes i s presented i n parti al ful fi l ment o f the requi rements for the degree of Doctor o f
Phi l osophy i n B iochemi stry a t Mas sey Uni vers i ty, New Zeal and .
John Beresford SMART
1980
ii .
ABSTRACT
Pyru vate kinase ca tal yses 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 tha t pyruvate kinase activity is much higher than that of pyruvate , orthophosphate dikinase which catal yses 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 fo r biosyn thesis . The
regul ato ry properties of this enzyme were accordingly studied .
The partial l y 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 satura tion 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 gl ycol ytic 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 substra te . 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-maxima l inhibition was obtained at 1 . 8 mM ATP and 1 • 4 mM Pi . The inhibition of both Pi and ATP could be l argely overcome by G6P . The G6P activation and other regul atory properties of the enzyme were pH dependent .
On the bas i s of th i s i n vi tro study i t was suggested that the acti vi ty of pyruvate kinase i n v i vo i s de termi 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 su fficientl y high when cul ture s 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 glycol yti c i ntermedi ates were measured in P . s hermani 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
pre sence of h i gh l evel s of gl ucose or glycerol , G6P was present at l evel s between 5 and 16 mM i n the cel l .
F6P and FBP were always 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 cantl y higher 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 gl ycero 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 i ty , 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 substra tes 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 s hown to be present i n l actate-grown cel l s . The other inhi bi tor, Pi , mus t 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 l evel s of substrates (PEP and ADP) and effectors (G6P and ATP) found in the ce l l indicated tha t . at l evel s above 10 mM Pi• concentrations of 5-15 mM G6P
(as found in cel ls growing on gl ucose and gl ycerol) 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 negl igibl 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
l imited 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 gl ycol ysis .
I t is also proposed that G6P may be invol ved in partitioning g l ucose metabol ism via glycolysis 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 s pecul a tive .
ACKNOWLEDGEMENTS
I wish to thank my Supervisor, Dr G . G . Pri tchard , for his 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 i cal 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 his advi ce concerning the fl uorometri c analyses of metabol i tes and the Appl i ed B i ochemi stry Di vi sion of 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 s t of Contents li st of Fi gures l i st of Tables
li st o f Abbrevi ation s
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 reg ul ati ng
carbohydrate metabol i sm i n bacteri a
Ca rbohydrate metabol i sm i n propi on i c aci d bacteri a Aims of th i s i n vesti gati on
CHAPTER 2 - MATERIAL S AND METHODS 2 . 1
2 . 2 2 . 3 2 . 4
2 . 5
2 .6 2 . 7
Introduct i on 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 sus pens i ons
Estimati on of s ubstrate s and products i n the growth medi 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 mati on
Measurement of enzyme acti vi ti es
Page Number i i
V
vi xi i xiv 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 I BACTER I UM SHERMAN I 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 ki nases P . shermani i pyruvate ki nase Pyruvate ki 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 o f the puri fi cati on scheme Properties of the parti al ly puri fi ed pyruvate k i nase from P. s he nnani i
4 . 3 . 1 Pyruvate-ki nase assay
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 ne ti c data
E ffect of enzyme concentration on acti vi ty Stud i es 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 s aturation.curve
Rel ationshi p between pyruvate ki nase acti vi ty and varyi n g ADP concentrati on
4. 3 . 9 . 1
4. 3 . 9 . 2
Effect o f PEP concentrati on on the ADP saturation curve
E ffect of 2 . 0 mM G6P on the ADP saturation curve
Effect o f 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 o f G6P on Mg++ acti vation 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 ki nase
Effect of varyi ng G6 P concentration on pyruvate ki nase activi ty
Effect of varyi ng ATP concentration o n pyruvate ki nase acti vi ty
9 4
96 96
4.3 . 15 4. 3 . 16
4. 3. 17
4 . 3 . 18
4. 4 Di scussion
Effect of varyi ng P; concentration on pyruvate ki nase acti v i ty
I nteracti on between G6P and ATP or P;
i n the rel ati ons hi p between pyruvate ki nase acti vi ty and PEP concentration
E ffect of pH on the ki neti c and al l oster i c properties o f pyruvate ki nase
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
110
112
CHAPTER 5 - Jli V I VO LEVELS OF METABOL ITES IN P . SHE RMAN I I GROWI NG UNDER D I FFERENT NUTRITI ONAL CONDI T I ONS
5 . 1 5 . 2
5 . 3
I ntroducti on
Compari son of metabol i te l evel s in res ti 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 glycol yti c i nte rmedi ates
�vi vo concentrati ons of metabol i tes i n batch and conti nuo us c ul tures of P . shermani i
5 . 3 . 1
5 . 3 . 2
Compari son o f extraction procedures for analysi s of gl ycolyti c i ntermedi ates Batch cul ture stud i es
1 1 8 1 1 8
1 20 121
125
127
1 30 5 . 3 . 2 . 1 Prel i mi nary i nvesti gati on of i n vi vo 130
concentrati ons of metabol i tes
5 . 3 . 3
5 . 4 Di scussion
5 . 3 . 2 . 2 Detai l ed study of metabol i te l evel s on ba tc h c ul tures of P . sherman i i
Conti nuous c ul tures 5 . 3 . 3 . 1
5 . 3 . 3. 2
5 . 3 . 3. 3
Carbon bal ances d uri ng conti n uous growth on l actate , gl ucose and glycerol defi ned media
I n v i vo concentrati ons of meta bol i tes i n conti n uous cul tures Spec i f i c acti v i t i es of sel ected enzymes i n cont i n uo us cul tures
CHAPTER 6 - THE ROLE OF G6P I N THE REGULATI ON OF METABOLI SM OF P . SHERMAN I I
6 . 1 6 . 2
6 . 3
6 . 4
I ntroducti on
Rel ations hi 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 nves ti gati on of the regul ati on of pyruvate k i nase acti vi ty a t i n vi vo concentrati ons of s ubs tra tes and e ffectors
6 . 3 . 1 6 . 3 . 2
Di scussion
Parti al puri fi cati on of pyruvate k i nase Studi es on the parti al ly p uri 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 acta te defi ned med i um
CHAPTER 8 - GENERAL DI SCU SSI ON 8 . 1
8 . 2
8.3
Regul ati on of pyruvate k i nase duri ng gl ycolys i s and gl uconeogenesi s
Further pos si bl e rol es of G6 P as a regul ator of carbohydra te metabol i sm in P.shermani i
Areas for further study Bi bl i ography
167 167
170 174
178
184
19 1 196
L I ST OF FIGURES
Fi gure Number Ti tl e
1 . 2 Carbohydrate metabol i sm i n P . s hermani i . 2 . 4 Rel ati onshi 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 . shermani i i n the defi ned medi um on lactate , gl ucose and glycerol .
Speci fi c acti vi ty of pyruvate ki nase d uri ng growth of P . s hermani i .
DEAE - Sephadex i on exchange chroma tography.
Sephacryl S200 gel fi l tration . Treatment of ki neti c data .
Effect of enzyme concentrati on on acti vi ty . Stabi l i ty o f pyruvate k i nase .
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 ons h i p between acti vi ty and PEP concentrati on .
Effect of PEP on the rel ati ons h 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 ons h i p between pyruvate ki nase acti vi ty and ADP
concentration .
Effect of MnCl 2 concen tration 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
Effect of G6P concentrati on on pyruvate ki nase act i v i ty.
Effect of ATP concentrati on on pyruvate ki nase acti vi ty.
Effect of Pi concentrati on on pyruvate ki 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 ki 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 acta te ' concentrati on.
Pyruvate ki nase acti vi ty at in v i vo concentrati ons of subs trates and effectors .
Rel ations hi p between gl ucose 6-phosphate dehydrogenase act i v 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 .
Summa ry 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 pyruva te 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 i onshi 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 hermani i con ti nuous cul tures .
I n vi vo l evel s of me tabol i tes i n continuous cul tures .
135
139
142
Enzyme l evel 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 ati ons hi p be tween external glycerol concen- 156 trati on and i n ternal G6P and F6P concentra tions .
Rel ati onshi p between external l actate concentr- 159 ati on and i nternal pyruvate concentration .
Partial puri fi cati on of pyruvate ki nase . 161 Effect of i norgani c phosphate on the cooperati vi ty 164 of G6P acti vati on of pyruva te k i nas e .
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
glycerol 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
di ethyl ami noethyl -
di hydroxyacetone phosphate deoxyri bonucl ease
Entner-Doudoroff
ethyl enedi ami ne tetra-aceti c aci d Embden -Meyerhof-Parnas
fructose 1 ,6-bi sphosphate fructose 6-phos pha te
J
glyceral dehyde 3-phosphate )guanos i ne 5 ' -di phosphate
glycerol 3-phosphate , a-gl ycerophosphate gl ucose 6-phosphate
guanosi ne 5 ' -tri phosphate
N-2-hydroxyethyl pi perazi ne-N ' -2-ethanesul phon i c a ci d hexose monophos phate
i nosi ne 5 ' -di phosphate
2�N-morphol i no_lethane sul phoni c aci d ni coti nami de adeni ne dinucl eotide
ni coti namide adeni ne dinucl eoti de , reduced
NADP NADPH nH 0°540 O AA 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
n i coti nami de adeni ne di nucl eotide phosphate
ni 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 -phos phogl uconate 3-phosphoglyceri c aci d i norgani c phosphate i norgani c pyrophos phate 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
maxi mum vel oci ty
Escheri ch i a col i Neurospora cras sa Streptococcus l a cti s
