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Activation and inhibition of the phosphoglycerate kinase reaction by ATP4−
283 Biochimica et Biophysica Acta, 481 (1977) 283--287 © Elsevier/North-Holland Biomedical P r e s s
BBA R e p o r t BBA 61317
ACTIVATION AND INHIBITION OF THE P H O S P H O G L Y C E R A T E KINASE REACTION BY ATP*-
M ~ R T H A LARSSON-RAZNIKIEWICZ* and BIRGITTA 8CHIERBECK
Department of Biochemistry, University of G~'teborg and Chalmers Institute of Technology, Fack, S-402 20 Go'teborg 5, and Department of Chemistry, Agricultural College of Sweden, S-750 07 Uppsala 7 (Sweden) (Received November 12th, 1976)
Summary A kinetic study for understanding h o w the phosphoglycerate kinase (ATP: 3-phospho-Dglycerate 1-phosphotransferase, EC 2.7.2.3) reaction discriminates between MgATP 2- and ATP 4- is presented. The results show that in contrast to MgATP 2-, ATP 4- is competitive with 3-phospho-D-glycerate. ATP 4- binds to the free enzyme as an inhibitor. When binding to the enzyme-MgATp2--(3-phospho-D-glycerate) complex, ATP 4- acts as an activator.
Phosphoglycerate kinase (EC 2.7.2.3) catalyses the reversible transfer of a phosphoryl group from MgATP 2- to 3-P-glycerate. The substrates are capable of binding to the enzyme independently, and a rapid equilibrium random mechanism fits the available data [1--5]. At elevated concentrations either substrate causes activation probably by binding to a second site in the protein molecule [1]. The ability of the enzyme molecule to bind two molecules of either substrate has been shown by equilibrium studies [6,7]. MgATP 2- is the true substrate in the catalytic reaction [8]. After it became evident [2] that MgATP 2- and ADP 3- preferentially bind to different sites, the obvious question was: Does the metal ion steer the nucleotide to a specific site in the enzyme? Phosphoglycerate kinase was prepared from baker's yeast by a m e t h o d described earlier [9] and the main electrophoretic c o m p o n e n t 2 was used. The reagents for determining the activity were from Sigma Chemical Co. *To w h o m correspondence should be addressed. Permanent address: D e p a r t m e n t of Chemistry, Agricultural College of Sweden, S-750 07 Uppsala 7, Sweden.
284
Only analytical grade reagents were used and all solutions were made with glass-distilled water. Contaminating metal ions were removed as described earlier [8]. The activity of phosphoglycerate kinase was measured spectrophotometrically [10] and expressed as the initial velocity, v (dA36Jdt)t= o in min -I (cf. ref, 8), The assay mixture contained 50 mM Tris. HCI buffer (pH 7.80 at 25 ° C) and 0.5 mM NADH in addition to the reagents mentioned in the text. About 0.2 U of phosphoglycerate kinase and 60 U of glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) were used per ml of the assay mixture. For estimation of the concentrations of ATP 4-, MgATP 2- and Mg2+, the following dissociation constants were used: MgATP 2-, 0.08 mM (cf. ref. 2) and 3-P-glycerate-Mg2+, 10 mM [11]. Effects of ATI~- on the phosphoglycerate kinase activity is illustrated in Fig. 1. ATP 4- up to about 1 mM appears as an activator. At higher concentrations inhibition becomes more and more predominant. A considerable change in ionic strength occurs with the increase in ATP 4- concentration. As seen in Fig. 1 its possible effect on the activity appears negligible. Figs. 2 and 3 show that ATP 4- is a competitive inhibitor of both the substrates. The secondary plots derived from these figures have a parabolic form (inset plots), indicating that there are at least two inhibitor molecules binding simultaneously to the enzyme. Assuming that the ATP 4- molecules are bound in sequence to two different sites in the free enzyme, the rate of the inhibited reaction can be described as: =
1 1 v=~
tl
K_.A_ KB ( 1 + [ I ] + [I]2 " K A K B I + [A] + [B] + K-~ K--~I j [A] [B]
r
I
(1)
I
0.5
0.4
"~" c
0.3
e
0.2
0.t
o
I'o [.Tp 4-]
l's
2'o
(,,,.)
Fig. 1. O v e r a l l e f f e c t s o f A T p 4 - o n the p h o s p h o g l y c e r a t e kinase activity. T h e e x p e r i m e n t w a s p e r f o r m e d at a c o n s t a n t KCI c o n c e n t r a t i o n o f 0 . 2 5 M (A). A successive increase i n i o n i c s t r e n g t h ( 0 . 1 9 M as a m a x i m u m i n c r e a s e ) o c c u r r e d w i t h increasing A T P 4 - e o n c e n t r a t i o n s . T h e KC1 c o n c e n t r a t i o n w a s v a r i e d ( o ) t o c o u n t e r a c t t h e c o r r e s p o n d i n g ionic strength effect. The S-P-glycerate c o n c e n t r a t i o n w a s 2 m M a n d t h e M g A T P 2 - c o n c e n t r a t i o n 1 raM.
285
,o
/ 2~o°
3O
°
[ATP 4-] (mM)
¢ v
/
E
D --
v
20
10
I
I
I
[
I
)
5 1 / [MgATP 2"]
I
I
1|0
]
(mM -1)
Fig. 2. E f f e c t s o f A T P 4 " - o n t h e p h o s p h o g l y c e r a t e k i n a s e a c t i v i t y a t v a r y i n g M g A T P 2 - c o n c e n t r a t i o n s . T h e A T p 4 - c o n c e n t r a t i n n s w e r e o, 3 r a M ; ~, 5 m M ; V, 7 m M ; o, I 0 m M ; O, 1 6 m M . T h e 3 - P - g l y c e r a t e c o n c e n t r a t i o n w a s 2 raM. T h e i n s e t f i g u r e s h o w s t h e s l o p e o f t h e L i n e w e a v e r - B u r k p l o t s as a f u n c t i o n of the ATp4-concentration. The points represent the slopes experimentally obtained and the solid line t h e e q u a t i o n o f t h e s l o p e e s t i m a t e d f r o m E q n . 1, w i t h t h e i n h i b i t o r c o n s t a n t s d e t e r m i n e d (cf. t h e t e x t ) .
.
,oo
.
.
.
L 12
'
'
'
,
,
,
lo
[ATP
'
/° '
'
/-
/
5
3
/
4-](raM)
do ~
j
/
20
"
60
40
v
20
5 1 / [3-P-GLYCERATE]
10 Cram- I )
Fig. 3. E f f e c t s o f A T p 4 - o n t h e a c t i v i t y o f p h o s p h o g i y c e r a t e k i n a s e a t v a r y i n g c o n c e n t r a t i o n s o f f r e e 3 - P - g l y c e r a t e . T h e A T P ~- c o n c e n t r a t i o n s a n d s y m b o l s a r e as i n Fig. 2. T h e M g A T P 2- c o n c e n t r a t i o n w a s 1 m M , T h e i n s e t f i g u r e s h o w s t h e s l o p e o f t h e L i n e w e a v e r - B u r k p l o t s as a f u n c t i o n o f A T P *~-. T h e p o i n t s r e p r e s e n t t h e s l o p e s e x p e r i m e n t a l l y o b t a i n e d a n d t h e s o l i d line t h e e q u a t i o n o f t h e s l o p e e s t i m a t e d f r o m E q n . 1, w i t h t h e i n h i b i t o r c o n s t a n t s d e t e r m i n e d (cf. t h e t e x t ) .
286 A symbolizes MgATP 2- and B 3-P-glycerate. KA and KB are the correspondingMichaelis constants, 0.5 mM and 0.62 mM, respectively [1]. ATP 4- is represented by I and its inhibitor constants by Ki and K~. The slope of both curves in the insets of Figs. 2 and 3 was plotted as a function of the ATP 4concentration. The two straight lines obtained were then used to calculate the inhibitor constants. Ki and K~ equal to 0.92 mM and 16 mM, respectively, fit the data of Fig. 2. The corresponding values from the results in Fig. 3 are 1.1 mM and 17 mM. In the calculations no consideration has been taken to the activation observed at low ATP 4- concentrations. Multiple binding sites for ATP 4- in the enzyme are required to explain the phenomena of activation and inhibition presented in Fig. I as well as the parabolic secondary plots shown in Figs. 2 and 3. Either substrate is able to nullify the inhibition. Thus ATP 4- competes with the substrates for the free form of the enzyme. LnATP- has earlier been shown to behave very much as ATP 4-, when related to the MgATP 2- kinetics [7]. It now appears possible to discriminate between different alternatives [6] of binding two ATP 4- molecules to the enzyme. X-ray crystallographic results [12] show that it is improbable that the binding sites are equivalent. The absence of a non-competitive contribution in the inhibition patterns would rule out the possibility that the binding sites are independent. It seems very likely that binding in sequence is the correct alternative. These conclusions agree with the earlier suggestions on 3-P-glycerate binding to the enzyme [6]. The inhibitor constants estimated from data presented in Figs. 2 and 3 were compared with the dissociation constants obtained from equilibrium studies [6], assuming ATP 4- binding in a compulsory order. Ki agrees nicely with the corresponding dissociation constant. K~ appears to be twice as large, a p h e n o m e n o n to be explained by the activation that has not been considered in Eqn. 1. Our results suggest that ATP 4- binding as an activator occurs only when the catalytic site is occupied by MgATP 2- and/or 3-P-glycerate. ATP 4binding as an inhibitor occurs only to the free form of the enzyme. Neither MgATP 2- nor 3-P-glycerate was present in the system used in the studies on binding of AT1~ - by equilibrium dialysis [6]. Simultaneous binding of ATP 4- and 3-P-glycerate to the active centre appears to be impossible, supporting the earlier suggestion that MgATP 2- is the true form of the nucleotide substrate [8]. Thus, complex formation with the divalent metal ion prior to binding to the active centre is necessary in case ATP is needed to form a catalytically functional enzyme-substrate complex. Some interaction appears to occur between the catalytic centre and an additional nucleotide binding site, offering possibilities of regulating the catalytic reaction. Work in progress will further discuss that phenomenon. We wish to thank Professor Bo G. Malmstrom for his continuous support of this investigation and Mrs. Ingrid Niith for excellent technical assistance. This work was supported by research grants from the Swedish Natural Science Research Council. A predoctoral fellowship from Stiftelsen Bengt Lundqvists Minne to one of us (B.S.) is also gratefully acknowledged.
287
References 1 2 3 4 5 6 7 8 9 10 11 12
Larsson-Ra~nikiewicz, M. (1967) Biochim, Biophys. Acta 132, 33--40 Larsson-Ra~nikiewicz, M. and Arvidsson, L. (1971) Eur. J. Biochem. 22, 506--512 Roustan, C., Brevet, A., Pradel, L.-A. and van Thoai, N. (1973) Eur. J. Biochem. 3 7 , 2 4 8 - - 2 5 5 Janson, C.A. and Cleland, W.W. (1974) J. Biol. Chem. 249, 2567--2571 Larsson-Ra~nikiewicz, M. and Schierbeck, B. (1974) Biochem. Biophys. Res. C ommun. 57, 627- - 634 Larsson-Ra~nikiewicz, M. (1973) Arch. Biochem. Biophys. 158, 754--762 Tansweil, P., Westhead, E.W. and Williams, R.J.P. (1974) FEBS Lett. 48, 60--63 Larsson-Ra~nikiewicz, M. (1964) Biochim. Biophys. Acta 85, 60--68 Arvidsson, L., Schierheck, B. and Larsson-Ra~nikiewiez, M. (1976) Acta Chem. Scand. B30, 228--234 Bucher, Th. (1947) Biochim. Biophys. Acta 1 , 2 9 2 - - 3 1 4 Larsson-Ra~nikiewicz, M. (1972) Eur. J. Biochem. 30, 579--583 Bryant, T.N., Watson, H.C. and Wendell, P.L. (1974) Nature 247, 14--17
BBA R e p o r t BBA 61317
ACTIVATION AND INHIBITION OF THE P H O S P H O G L Y C E R A T E KINASE REACTION BY ATP*-
M ~ R T H A LARSSON-RAZNIKIEWICZ* and BIRGITTA 8CHIERBECK
Department of Biochemistry, University of G~'teborg and Chalmers Institute of Technology, Fack, S-402 20 Go'teborg 5, and Department of Chemistry, Agricultural College of Sweden, S-750 07 Uppsala 7 (Sweden) (Received November 12th, 1976)
Summary A kinetic study for understanding h o w the phosphoglycerate kinase (ATP: 3-phospho-Dglycerate 1-phosphotransferase, EC 2.7.2.3) reaction discriminates between MgATP 2- and ATP 4- is presented. The results show that in contrast to MgATP 2-, ATP 4- is competitive with 3-phospho-D-glycerate. ATP 4- binds to the free enzyme as an inhibitor. When binding to the enzyme-MgATp2--(3-phospho-D-glycerate) complex, ATP 4- acts as an activator.
Phosphoglycerate kinase (EC 2.7.2.3) catalyses the reversible transfer of a phosphoryl group from MgATP 2- to 3-P-glycerate. The substrates are capable of binding to the enzyme independently, and a rapid equilibrium random mechanism fits the available data [1--5]. At elevated concentrations either substrate causes activation probably by binding to a second site in the protein molecule [1]. The ability of the enzyme molecule to bind two molecules of either substrate has been shown by equilibrium studies [6,7]. MgATP 2- is the true substrate in the catalytic reaction [8]. After it became evident [2] that MgATP 2- and ADP 3- preferentially bind to different sites, the obvious question was: Does the metal ion steer the nucleotide to a specific site in the enzyme? Phosphoglycerate kinase was prepared from baker's yeast by a m e t h o d described earlier [9] and the main electrophoretic c o m p o n e n t 2 was used. The reagents for determining the activity were from Sigma Chemical Co. *To w h o m correspondence should be addressed. Permanent address: D e p a r t m e n t of Chemistry, Agricultural College of Sweden, S-750 07 Uppsala 7, Sweden.
284
Only analytical grade reagents were used and all solutions were made with glass-distilled water. Contaminating metal ions were removed as described earlier [8]. The activity of phosphoglycerate kinase was measured spectrophotometrically [10] and expressed as the initial velocity, v (dA36Jdt)t= o in min -I (cf. ref, 8), The assay mixture contained 50 mM Tris. HCI buffer (pH 7.80 at 25 ° C) and 0.5 mM NADH in addition to the reagents mentioned in the text. About 0.2 U of phosphoglycerate kinase and 60 U of glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) were used per ml of the assay mixture. For estimation of the concentrations of ATP 4-, MgATP 2- and Mg2+, the following dissociation constants were used: MgATP 2-, 0.08 mM (cf. ref. 2) and 3-P-glycerate-Mg2+, 10 mM [11]. Effects of ATI~- on the phosphoglycerate kinase activity is illustrated in Fig. 1. ATP 4- up to about 1 mM appears as an activator. At higher concentrations inhibition becomes more and more predominant. A considerable change in ionic strength occurs with the increase in ATP 4- concentration. As seen in Fig. 1 its possible effect on the activity appears negligible. Figs. 2 and 3 show that ATP 4- is a competitive inhibitor of both the substrates. The secondary plots derived from these figures have a parabolic form (inset plots), indicating that there are at least two inhibitor molecules binding simultaneously to the enzyme. Assuming that the ATP 4- molecules are bound in sequence to two different sites in the free enzyme, the rate of the inhibited reaction can be described as: =
1 1 v=~
tl
K_.A_ KB ( 1 + [ I ] + [I]2 " K A K B I + [A] + [B] + K-~ K--~I j [A] [B]
r
I
(1)
I
0.5
0.4
"~" c
0.3
e
0.2
0.t
o
I'o [.Tp 4-]
l's
2'o
(,,,.)
Fig. 1. O v e r a l l e f f e c t s o f A T p 4 - o n the p h o s p h o g l y c e r a t e kinase activity. T h e e x p e r i m e n t w a s p e r f o r m e d at a c o n s t a n t KCI c o n c e n t r a t i o n o f 0 . 2 5 M (A). A successive increase i n i o n i c s t r e n g t h ( 0 . 1 9 M as a m a x i m u m i n c r e a s e ) o c c u r r e d w i t h increasing A T P 4 - e o n c e n t r a t i o n s . T h e KC1 c o n c e n t r a t i o n w a s v a r i e d ( o ) t o c o u n t e r a c t t h e c o r r e s p o n d i n g ionic strength effect. The S-P-glycerate c o n c e n t r a t i o n w a s 2 m M a n d t h e M g A T P 2 - c o n c e n t r a t i o n 1 raM.
285
,o
/ 2~o°
3O
°
[ATP 4-] (mM)
¢ v
/
E
D --
v
20
10
I
I
I
[
I
)
5 1 / [MgATP 2"]
I
I
1|0
]
(mM -1)
Fig. 2. E f f e c t s o f A T P 4 " - o n t h e p h o s p h o g l y c e r a t e k i n a s e a c t i v i t y a t v a r y i n g M g A T P 2 - c o n c e n t r a t i o n s . T h e A T p 4 - c o n c e n t r a t i n n s w e r e o, 3 r a M ; ~, 5 m M ; V, 7 m M ; o, I 0 m M ; O, 1 6 m M . T h e 3 - P - g l y c e r a t e c o n c e n t r a t i o n w a s 2 raM. T h e i n s e t f i g u r e s h o w s t h e s l o p e o f t h e L i n e w e a v e r - B u r k p l o t s as a f u n c t i o n of the ATp4-concentration. The points represent the slopes experimentally obtained and the solid line t h e e q u a t i o n o f t h e s l o p e e s t i m a t e d f r o m E q n . 1, w i t h t h e i n h i b i t o r c o n s t a n t s d e t e r m i n e d (cf. t h e t e x t ) .
.
,oo
.
.
.
L 12
'
'
'
,
,
,
lo
[ATP
'
/° '
'
/-
/
5
3
/
4-](raM)
do ~
j
/
20
"
60
40
v
20
5 1 / [3-P-GLYCERATE]
10 Cram- I )
Fig. 3. E f f e c t s o f A T p 4 - o n t h e a c t i v i t y o f p h o s p h o g i y c e r a t e k i n a s e a t v a r y i n g c o n c e n t r a t i o n s o f f r e e 3 - P - g l y c e r a t e . T h e A T P ~- c o n c e n t r a t i o n s a n d s y m b o l s a r e as i n Fig. 2. T h e M g A T P 2- c o n c e n t r a t i o n w a s 1 m M , T h e i n s e t f i g u r e s h o w s t h e s l o p e o f t h e L i n e w e a v e r - B u r k p l o t s as a f u n c t i o n o f A T P *~-. T h e p o i n t s r e p r e s e n t t h e s l o p e s e x p e r i m e n t a l l y o b t a i n e d a n d t h e s o l i d line t h e e q u a t i o n o f t h e s l o p e e s t i m a t e d f r o m E q n . 1, w i t h t h e i n h i b i t o r c o n s t a n t s d e t e r m i n e d (cf. t h e t e x t ) .
286 A symbolizes MgATP 2- and B 3-P-glycerate. KA and KB are the correspondingMichaelis constants, 0.5 mM and 0.62 mM, respectively [1]. ATP 4- is represented by I and its inhibitor constants by Ki and K~. The slope of both curves in the insets of Figs. 2 and 3 was plotted as a function of the ATP 4concentration. The two straight lines obtained were then used to calculate the inhibitor constants. Ki and K~ equal to 0.92 mM and 16 mM, respectively, fit the data of Fig. 2. The corresponding values from the results in Fig. 3 are 1.1 mM and 17 mM. In the calculations no consideration has been taken to the activation observed at low ATP 4- concentrations. Multiple binding sites for ATP 4- in the enzyme are required to explain the phenomena of activation and inhibition presented in Fig. I as well as the parabolic secondary plots shown in Figs. 2 and 3. Either substrate is able to nullify the inhibition. Thus ATP 4- competes with the substrates for the free form of the enzyme. LnATP- has earlier been shown to behave very much as ATP 4-, when related to the MgATP 2- kinetics [7]. It now appears possible to discriminate between different alternatives [6] of binding two ATP 4- molecules to the enzyme. X-ray crystallographic results [12] show that it is improbable that the binding sites are equivalent. The absence of a non-competitive contribution in the inhibition patterns would rule out the possibility that the binding sites are independent. It seems very likely that binding in sequence is the correct alternative. These conclusions agree with the earlier suggestions on 3-P-glycerate binding to the enzyme [6]. The inhibitor constants estimated from data presented in Figs. 2 and 3 were compared with the dissociation constants obtained from equilibrium studies [6], assuming ATP 4- binding in a compulsory order. Ki agrees nicely with the corresponding dissociation constant. K~ appears to be twice as large, a p h e n o m e n o n to be explained by the activation that has not been considered in Eqn. 1. Our results suggest that ATP 4- binding as an activator occurs only when the catalytic site is occupied by MgATP 2- and/or 3-P-glycerate. ATP 4binding as an inhibitor occurs only to the free form of the enzyme. Neither MgATP 2- nor 3-P-glycerate was present in the system used in the studies on binding of AT1~ - by equilibrium dialysis [6]. Simultaneous binding of ATP 4- and 3-P-glycerate to the active centre appears to be impossible, supporting the earlier suggestion that MgATP 2- is the true form of the nucleotide substrate [8]. Thus, complex formation with the divalent metal ion prior to binding to the active centre is necessary in case ATP is needed to form a catalytically functional enzyme-substrate complex. Some interaction appears to occur between the catalytic centre and an additional nucleotide binding site, offering possibilities of regulating the catalytic reaction. Work in progress will further discuss that phenomenon. We wish to thank Professor Bo G. Malmstrom for his continuous support of this investigation and Mrs. Ingrid Niith for excellent technical assistance. This work was supported by research grants from the Swedish Natural Science Research Council. A predoctoral fellowship from Stiftelsen Bengt Lundqvists Minne to one of us (B.S.) is also gratefully acknowledged.
287
References 1 2 3 4 5 6 7 8 9 10 11 12
Larsson-Ra~nikiewicz, M. (1967) Biochim, Biophys. Acta 132, 33--40 Larsson-Ra~nikiewicz, M. and Arvidsson, L. (1971) Eur. J. Biochem. 22, 506--512 Roustan, C., Brevet, A., Pradel, L.-A. and van Thoai, N. (1973) Eur. J. Biochem. 3 7 , 2 4 8 - - 2 5 5 Janson, C.A. and Cleland, W.W. (1974) J. Biol. Chem. 249, 2567--2571 Larsson-Ra~nikiewicz, M. and Schierbeck, B. (1974) Biochem. Biophys. Res. C ommun. 57, 627- - 634 Larsson-Ra~nikiewicz, M. (1973) Arch. Biochem. Biophys. 158, 754--762 Tansweil, P., Westhead, E.W. and Williams, R.J.P. (1974) FEBS Lett. 48, 60--63 Larsson-Ra~nikiewicz, M. (1964) Biochim. Biophys. Acta 85, 60--68 Arvidsson, L., Schierheck, B. and Larsson-Ra~nikiewiez, M. (1976) Acta Chem. Scand. B30, 228--234 Bucher, Th. (1947) Biochim. Biophys. Acta 1 , 2 9 2 - - 3 1 4 Larsson-Ra~nikiewicz, M. (1972) Eur. J. Biochem. 30, 579--583 Bryant, T.N., Watson, H.C. and Wendell, P.L. (1974) Nature 247, 14--17