119
Biochimica et Biophysica Acta, 438 (1976) 119--130
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 67837
THE P R O T O N T R A N S F E R REACTIONS CATALYZED BY YEAST P Y R U V A T E KINASE
S H A R O N R . F O R D a n d J A M E S L. R O B I N S O N
Division of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Ill. 61801 (U.S.A.)
(Received November 24th, 1975) Summary 1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10--100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction o f pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase {Robinson, J.L. and Rose, I.A. {1972) J. Biol. Chem. 247, 1096--1105). The pH-profiles and the divalent cation dependencies were similar for Fru1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization b y yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization b y the muscle enzyme which proceeds when ATP is replaced b y Pi or other dianions. 3. Fructose-l,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme. Introduction Pyruvate kinases (EC 2.7.1.40) display interspecies and intertissue differences in their regulatory properties [1,2]. Rabbit muscle pyruvate kinase obeys
l~uffers used through this study were: MES between pH 5.8 and 6.9, TES from pH 6.9 to 7.9, TAPS from pH 7.9 to 9.4 and glycine from pH 9.4 to 10.0. All buffers were adjusted to the indicated pH w i t h t e t r a m e t h y l a m m o n i u m h y d r o x i d e unless otherwise indicated. Abbreviations: ADP-CH2-P, 13,7ometbylene-adenosine triphospbate~ AMP-CH2-P, ~,~3-methyleneadenosine diphosphate~ MES, 2(N-morpholino)ethane sulfonate; TES, N -t ri s (hydroxyme t hyl )methy1-2-amino ethane sulfonate; TAPS, t r i s ( h y d r o x y m e t h y l ) m e t h y l a m i n o p r o p a n e sulfonate.
120
Michaelis-Menten kinetics while yeast pyruvate kinase exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and is allosterically activated by fructose-l,6-bisphosphate [3,4]. It is of interest to compare the activities of these t w o pyruvate kinases to see what elements of reactivity have been maintained throughout their evolution. The primary, physiologically important reaction catalyzed by pyruvate kinase is the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP. The overall reaction involves t w o elements: the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, and the transfer of a proton from water to form the methyl group of pyruvate. The equilibrium of the reaction is strongly pH20) would be expected. The observed isotope effect of 4--6 suggests that C-H bond breaking is not solely rate-limiting, b u t is not completely independent of the rate-limiting step [19]. One of the aims of this study was to compare the proton-transfer reactions of yeast pyruvate kinase with those that have previously been reported for muscle pyruvate kinase [10]. Yeast pyruvate kinase differs from the muscle enzyme in that the yeast enzyme shows cooperative phosphoenolpyruvate binding and allosteric activation by Fru-l,6-P2 [3,4]. This study shows that the parameters of the p r o t o n transfer reactions of yeast pyruvate kinase in the presence o f 1 mM Fru-l,6-P2 (which results in Michaelis-Menten kinetics) are very similar to those of muscle pyruvate kinase. The dependence of R w o n pH and divalent cation are very similar, although the absolute values with Mg 2÷ are lower for the yeast enzyme than for the muscle enzyme. In both c a s e s R w is low at low pH and increases dramatically with increasing pH. The pH dependence of R w for Co2+-activated yeast pyruvate kinase and muscle pyruvate kinase is similar to that observed with Mn 2+ and Mg 2+ as the divalent cation (data not shown). Since R w is a measure of the ratio of exchange of tritium from pyruvate to water compared to the release of pyruvate from the enzyme ( R w = /z_3/k4 in Scheme 1), these results mean the relative amount of pyruvate enolization that occurs, compared to the release of products, increases with increasing pH and is somewhat dependent on the divalent cation. Very low R w values were observed in the absence of Fru-l,6-P2 for all divalent cation investigated, suggesting that it plays a crucial role in allowing significant reversal of the proton transfer step at elevated pH. The role of Fru-l,6-P2 will be considered in more detail later. The dependence on pH and divalent cation of the rate of yeast pyruvate kinase-catalyzed pyruvate enolization (Vxw) is alSO very similar (in the presence of Fru-l,6-P2) to that observed for the muscle e n z y m e , vxw in the presence of Mn 2+ rises at slightly higher pH with the yeast enzyme and does not show the plateau observed with the muscle enzyme around pH 8 [10]. The pH-profile for the Mg 2+-enzyme is virtually identical for the two enzymes. The pH-profile for Co2+-yeast pyruvate kinase shows very low Vxw values with a maximum of 1.2 umol/min per mg at pH 7.4 (data not shown). The maximum V~T for Co 2+activated muscle pyruvate kinase is also a b o u t pH 7.4, b u t the rate is approximately 10-fold the rate for the yeast enzyme. The requirements for pyruvate enolization catalyzed by yeast pyruvate kinase were shown to be more stringent than for the muscle enzyme. Whereas inorganic phosphate or a series o f anions were able to replace ATP to p r o m o t e pyruvate enolization by muscle pyruvate kinase [9], there is a strict requirement for ATP in the yeast pyruvate kinase catalyzed enolization reaction. This strict requirement is observed in spite of the fact that phosphoryl transfer from ATP cannot be required for pyruvate enolization due to the differences in the
129 relative rates o f the t w o reactions. The fact that neither Pi, nor ADP, nor ADPCH2-P could replace ATP in this function suggests a strict steric requirement for ATP in the pyruvate enolization reaction. Thus, although the mechanism by which yeast pyruvate kinase catalyzes its physiologically important reaction is similar to the mechanism of rabbit muscle pyruvate kinase, the yeast enzyme appears more stringent in its substrate requirements. Another goal of this study was to investigate the role of Fru-l,6-P2 in the activity o f yeast pyruvate kinase. A review of the effect of Fru-l,6-P: on different parameters of enzyme activity (Table V) reveals that Fru-l,6-P: must be involved in the catalytic steps in addition to substrate-binding steps. When Mg 2+ is the divalent cation, Fru-l,6-P2 has a large effect on all parameters measured (except V of the forward reaction). Fru-l,6-P2 is involved in substrate binding, as evidenced b y its effect on S0.s for phosphoenolpyruvate. The effect of Fru1,6-P2 on RT and Vxw implies involvement at other steps in the mechanism. Fru-l,6-P2 could increase R w b y increasing the rate of pyruvate enolization (step --3) and/or b y decreasing the rate of p r o d u c t release from the enzyme (step +4). The effect on Vxw also suggests that pyruvate binding (step --4) and an enolization (step --3) are accelerated by Fru-l,6-P2. Since it increases all the partial reactions in the presence of Mg 2÷, one cannot determine from these data at which steps the Fru-l,6-P2 is most active. The effects of Fru-l,6-P2 on Mn2÷-activated yeast pyruvate kinase yield more information. In this case it has no effect on binding of phosphoenolpyruvate as measured b y S0.s, b u t affects some of the partial reactions. Fru-l,6-P2 has a strong effect on RT and Vxp with the Mn:+-activated enzyme, but has only a small effect on V~T. Since any substrate binding effects of Fru-l,6-P2 should affect V~T and Vxp equally, it is clear that at least part of the effect is on a catalytic step of the reaction. In summary, Fru-l,6-P: is involved in the binding of substrates to yeast pyruvate kinase and in addition affects the rates of the catalytic steps of the enzyme. When Mg 2÷ is the divalent cation, Fru-l,6-P2 greatly enhanced all measured reactions. When Mn 2÷ is used, some steps become much less sensitive to Fru-l,6-P2 activation; the binding of substrates and the enolization of pyruvate are largely independent of Fru-l,6-P2 under these conditions. Similarly, the flu-
TABLE V SUMMARY OF THE E F F E C T S OF FRU-1,6-P 2 ON Y E A S T P Y R U V A T E K I N A S E A C T I V I T Y Parameter
Effect of Fru-l,6-P 2 with Mg 2+ as d i v a l e n t c a t i o n
Mn 2+ as divalent c a t i o n no effect
Vforward *
no effect
SO. 5 *
large d e c r e a s e
small decrease
RT
large i n c r e a s e
large increase
VxT
large increase
s m a l l increase
Vxp
large i n c r e a s e
large increase
* D a t a f r o m L e B l o n d and R o b i n s o n [ 1 1 ] ; 8 0 . 5 is w i t h r e s p e c t t o p h o s p h o e n o l p y r u v a t e .
130 orokinase and hydroxylamine kinase reactions are shown to be little affected by the presence of Fru-l,6-P2 when Mn 2÷ is the activating divalent cation [ 1 1 ] . Since the sensitivity to Fru-l,6-P2 appears to depend on the concentration of Mn 2÷, it is reasonable to suggest that Mn 2÷ activates the partial reactions in a manner that cannot be accomplished with Me 2÷ Acknowledgments The authors thank D.J. LeBlond for isolating the yeast pyruvate kinase, P.J. Taylor for synthesizing the [3-3H]pyruvate and [3-3H]phosphoenolpyruvate, and S. Rosenzweig for preparing the [7.32p] ATP. Special thanks are due to W.T. Ford for assisting with the NMR experiments. The authors thank the Illinois Agricultural Experiment Station for support of their research. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Carbonell, J., Feliu, J.E., Marco, R. and Sols, A. (1973) EuL J. Biochem. 3 7 , 1 4 8 - - 1 5 6 Cardenas, J.M., Dyson, R.D. and Stxandholm, J.J. (1973) J. Biol. Chem. 248, 6931--6937 Hess, B., Haeckel, R. and Blank, K. (1966) Biochem. Biophys. Res. Commun. 24, 824--831 Hunsley, J.R. and Suelter, C.H. (1969) J. Biol. Chem. 244, 4 8 1 5 - - 4 8 2 2 McQuate, J.T. and Utter, M.F. (1959) J. Biol. Chem. 234, 2151--2157 Tietz, A. and Oehoa, S. (1958) Arch. Biochem. Biophys. 78, 477--493 Cottam, G.L., Kupieeki, F.P. and Coon, M.J. (1968) J. BioL Chem. 2 4 3 : 1 6 3 0 - - 1 6 3 7 Kayne, F.J. (1974) Biochem. Biophys. Res. Commun. 59, 8--12 Rose, I.A. (1960) J. Biol. Chem. 235, 1170--1177 Robinson, J.L. and Rose, LA. (1972) J. Biol. Chem. 247, 1 0 9 6 - - 1 1 0 5 LeBlond, D.J. and Robinson, J.L. (1976) Bioehem. Biophys. Acta, 4 3 8 , 1 0 8 - - 1 1 8 Aust, A., Yun, S.L. and Suelter, C.H. ( 1 9 7 5 ) i n Methods in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds.) Vol. 42, pp. 176--182, Academic Press, N e w York Meloche, H.P. (1970) Anal. Bioehem. 38, 389--392 Glynn, I.M. and Chappel], J.B. (1964) Biochem. J . 9 0 , 14 7--149 Anderson, L.E. and McClure, W.O. (1973) Anal. Biochem. 51, 173--179 Switzer, R.L. ( 1 9 6 9 ) J . Biol. Chem. 244, 2851--2863 Glascoe, P.K. and Long, F.A. (1960) J. Phys. Chem. 64, 188--190 Wieker, H.J. and Hess, B. (1971) Biochemistry 10, 1 2 4 3 - - 1 2 4 8 Northrop, D.B. (1975) Biochemistry 14, 2644--2651
Biochimica et Biophysica Acta, 438 (1976) 119--130
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 67837
THE P R O T O N T R A N S F E R REACTIONS CATALYZED BY YEAST P Y R U V A T E KINASE
S H A R O N R . F O R D a n d J A M E S L. R O B I N S O N
Division of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Ill. 61801 (U.S.A.)
(Received November 24th, 1975) Summary 1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10--100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction o f pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase {Robinson, J.L. and Rose, I.A. {1972) J. Biol. Chem. 247, 1096--1105). The pH-profiles and the divalent cation dependencies were similar for Fru1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization b y yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization b y the muscle enzyme which proceeds when ATP is replaced b y Pi or other dianions. 3. Fructose-l,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme. Introduction Pyruvate kinases (EC 2.7.1.40) display interspecies and intertissue differences in their regulatory properties [1,2]. Rabbit muscle pyruvate kinase obeys
l~uffers used through this study were: MES between pH 5.8 and 6.9, TES from pH 6.9 to 7.9, TAPS from pH 7.9 to 9.4 and glycine from pH 9.4 to 10.0. All buffers were adjusted to the indicated pH w i t h t e t r a m e t h y l a m m o n i u m h y d r o x i d e unless otherwise indicated. Abbreviations: ADP-CH2-P, 13,7ometbylene-adenosine triphospbate~ AMP-CH2-P, ~,~3-methyleneadenosine diphosphate~ MES, 2(N-morpholino)ethane sulfonate; TES, N -t ri s (hydroxyme t hyl )methy1-2-amino ethane sulfonate; TAPS, t r i s ( h y d r o x y m e t h y l ) m e t h y l a m i n o p r o p a n e sulfonate.
120
Michaelis-Menten kinetics while yeast pyruvate kinase exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and is allosterically activated by fructose-l,6-bisphosphate [3,4]. It is of interest to compare the activities of these t w o pyruvate kinases to see what elements of reactivity have been maintained throughout their evolution. The primary, physiologically important reaction catalyzed by pyruvate kinase is the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP. The overall reaction involves t w o elements: the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, and the transfer of a proton from water to form the methyl group of pyruvate. The equilibrium of the reaction is strongly pH20) would be expected. The observed isotope effect of 4--6 suggests that C-H bond breaking is not solely rate-limiting, b u t is not completely independent of the rate-limiting step [19]. One of the aims of this study was to compare the proton-transfer reactions of yeast pyruvate kinase with those that have previously been reported for muscle pyruvate kinase [10]. Yeast pyruvate kinase differs from the muscle enzyme in that the yeast enzyme shows cooperative phosphoenolpyruvate binding and allosteric activation by Fru-l,6-P2 [3,4]. This study shows that the parameters of the p r o t o n transfer reactions of yeast pyruvate kinase in the presence o f 1 mM Fru-l,6-P2 (which results in Michaelis-Menten kinetics) are very similar to those of muscle pyruvate kinase. The dependence of R w o n pH and divalent cation are very similar, although the absolute values with Mg 2÷ are lower for the yeast enzyme than for the muscle enzyme. In both c a s e s R w is low at low pH and increases dramatically with increasing pH. The pH dependence of R w for Co2+-activated yeast pyruvate kinase and muscle pyruvate kinase is similar to that observed with Mn 2+ and Mg 2+ as the divalent cation (data not shown). Since R w is a measure of the ratio of exchange of tritium from pyruvate to water compared to the release of pyruvate from the enzyme ( R w = /z_3/k4 in Scheme 1), these results mean the relative amount of pyruvate enolization that occurs, compared to the release of products, increases with increasing pH and is somewhat dependent on the divalent cation. Very low R w values were observed in the absence of Fru-l,6-P2 for all divalent cation investigated, suggesting that it plays a crucial role in allowing significant reversal of the proton transfer step at elevated pH. The role of Fru-l,6-P2 will be considered in more detail later. The dependence on pH and divalent cation of the rate of yeast pyruvate kinase-catalyzed pyruvate enolization (Vxw) is alSO very similar (in the presence of Fru-l,6-P2) to that observed for the muscle e n z y m e , vxw in the presence of Mn 2+ rises at slightly higher pH with the yeast enzyme and does not show the plateau observed with the muscle enzyme around pH 8 [10]. The pH-profile for the Mg 2+-enzyme is virtually identical for the two enzymes. The pH-profile for Co2+-yeast pyruvate kinase shows very low Vxw values with a maximum of 1.2 umol/min per mg at pH 7.4 (data not shown). The maximum V~T for Co 2+activated muscle pyruvate kinase is also a b o u t pH 7.4, b u t the rate is approximately 10-fold the rate for the yeast enzyme. The requirements for pyruvate enolization catalyzed by yeast pyruvate kinase were shown to be more stringent than for the muscle enzyme. Whereas inorganic phosphate or a series o f anions were able to replace ATP to p r o m o t e pyruvate enolization by muscle pyruvate kinase [9], there is a strict requirement for ATP in the yeast pyruvate kinase catalyzed enolization reaction. This strict requirement is observed in spite of the fact that phosphoryl transfer from ATP cannot be required for pyruvate enolization due to the differences in the
129 relative rates o f the t w o reactions. The fact that neither Pi, nor ADP, nor ADPCH2-P could replace ATP in this function suggests a strict steric requirement for ATP in the pyruvate enolization reaction. Thus, although the mechanism by which yeast pyruvate kinase catalyzes its physiologically important reaction is similar to the mechanism of rabbit muscle pyruvate kinase, the yeast enzyme appears more stringent in its substrate requirements. Another goal of this study was to investigate the role of Fru-l,6-P2 in the activity o f yeast pyruvate kinase. A review of the effect of Fru-l,6-P: on different parameters of enzyme activity (Table V) reveals that Fru-l,6-P: must be involved in the catalytic steps in addition to substrate-binding steps. When Mg 2+ is the divalent cation, Fru-l,6-P2 has a large effect on all parameters measured (except V of the forward reaction). Fru-l,6-P2 is involved in substrate binding, as evidenced b y its effect on S0.s for phosphoenolpyruvate. The effect of Fru1,6-P2 on RT and Vxw implies involvement at other steps in the mechanism. Fru-l,6-P2 could increase R w b y increasing the rate of pyruvate enolization (step --3) and/or b y decreasing the rate of p r o d u c t release from the enzyme (step +4). The effect on Vxw also suggests that pyruvate binding (step --4) and an enolization (step --3) are accelerated by Fru-l,6-P2. Since it increases all the partial reactions in the presence of Mg 2÷, one cannot determine from these data at which steps the Fru-l,6-P2 is most active. The effects of Fru-l,6-P2 on Mn2÷-activated yeast pyruvate kinase yield more information. In this case it has no effect on binding of phosphoenolpyruvate as measured b y S0.s, b u t affects some of the partial reactions. Fru-l,6-P2 has a strong effect on RT and Vxp with the Mn:+-activated enzyme, but has only a small effect on V~T. Since any substrate binding effects of Fru-l,6-P2 should affect V~T and Vxp equally, it is clear that at least part of the effect is on a catalytic step of the reaction. In summary, Fru-l,6-P: is involved in the binding of substrates to yeast pyruvate kinase and in addition affects the rates of the catalytic steps of the enzyme. When Mg 2÷ is the divalent cation, Fru-l,6-P2 greatly enhanced all measured reactions. When Mn 2÷ is used, some steps become much less sensitive to Fru-l,6-P2 activation; the binding of substrates and the enolization of pyruvate are largely independent of Fru-l,6-P2 under these conditions. Similarly, the flu-
TABLE V SUMMARY OF THE E F F E C T S OF FRU-1,6-P 2 ON Y E A S T P Y R U V A T E K I N A S E A C T I V I T Y Parameter
Effect of Fru-l,6-P 2 with Mg 2+ as d i v a l e n t c a t i o n
Mn 2+ as divalent c a t i o n no effect
Vforward *
no effect
SO. 5 *
large d e c r e a s e
small decrease
RT
large i n c r e a s e
large increase
VxT
large increase
s m a l l increase
Vxp
large i n c r e a s e
large increase
* D a t a f r o m L e B l o n d and R o b i n s o n [ 1 1 ] ; 8 0 . 5 is w i t h r e s p e c t t o p h o s p h o e n o l p y r u v a t e .
130 orokinase and hydroxylamine kinase reactions are shown to be little affected by the presence of Fru-l,6-P2 when Mn 2÷ is the activating divalent cation [ 1 1 ] . Since the sensitivity to Fru-l,6-P2 appears to depend on the concentration of Mn 2÷, it is reasonable to suggest that Mn 2÷ activates the partial reactions in a manner that cannot be accomplished with Me 2÷ Acknowledgments The authors thank D.J. LeBlond for isolating the yeast pyruvate kinase, P.J. Taylor for synthesizing the [3-3H]pyruvate and [3-3H]phosphoenolpyruvate, and S. Rosenzweig for preparing the [7.32p] ATP. Special thanks are due to W.T. Ford for assisting with the NMR experiments. The authors thank the Illinois Agricultural Experiment Station for support of their research. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Carbonell, J., Feliu, J.E., Marco, R. and Sols, A. (1973) EuL J. Biochem. 3 7 , 1 4 8 - - 1 5 6 Cardenas, J.M., Dyson, R.D. and Stxandholm, J.J. (1973) J. Biol. Chem. 248, 6931--6937 Hess, B., Haeckel, R. and Blank, K. (1966) Biochem. Biophys. Res. Commun. 24, 824--831 Hunsley, J.R. and Suelter, C.H. (1969) J. Biol. Chem. 244, 4 8 1 5 - - 4 8 2 2 McQuate, J.T. and Utter, M.F. (1959) J. Biol. Chem. 234, 2151--2157 Tietz, A. and Oehoa, S. (1958) Arch. Biochem. Biophys. 78, 477--493 Cottam, G.L., Kupieeki, F.P. and Coon, M.J. (1968) J. BioL Chem. 2 4 3 : 1 6 3 0 - - 1 6 3 7 Kayne, F.J. (1974) Biochem. Biophys. Res. Commun. 59, 8--12 Rose, I.A. (1960) J. Biol. Chem. 235, 1170--1177 Robinson, J.L. and Rose, LA. (1972) J. Biol. Chem. 247, 1 0 9 6 - - 1 1 0 5 LeBlond, D.J. and Robinson, J.L. (1976) Bioehem. Biophys. Acta, 4 3 8 , 1 0 8 - - 1 1 8 Aust, A., Yun, S.L. and Suelter, C.H. ( 1 9 7 5 ) i n Methods in E n z y m o l o g y (Colowick, S.P. and Kaplan, N.O., eds.) Vol. 42, pp. 176--182, Academic Press, N e w York Meloche, H.P. (1970) Anal. Bioehem. 38, 389--392 Glynn, I.M. and Chappel], J.B. (1964) Biochem. J . 9 0 , 14 7--149 Anderson, L.E. and McClure, W.O. (1973) Anal. Biochem. 51, 173--179 Switzer, R.L. ( 1 9 6 9 ) J . Biol. Chem. 244, 2851--2863 Glascoe, P.K. and Long, F.A. (1960) J. Phys. Chem. 64, 188--190 Wieker, H.J. and Hess, B. (1971) Biochemistry 10, 1 2 4 3 - - 1 2 4 8 Northrop, D.B. (1975) Biochemistry 14, 2644--2651
