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Comparative kinetic studies of Mn2+-activated and fructose-1,6-P-modified Mg2+-activated pyruvate kinase from Concholepas concholepas
Comp. Biochem. PhysioL Vol. 82B, No. 1, pp. 63-65, 1985
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE KINETIC STUDIES OF Mn 2+-ACTIVATED AND FRUCTOSE- 1,6-P-MODIFIED
Mg2÷-ACTIVATED PYRUVATE KINASE FROM CONCHOLEPAS CONCHOLEPAS NELSON CARVAJAL, RUBY GONZALEZ, ARSENIO MORAN and ANA MARIA OYARCE Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Concepci6n, Chile
(Received 11 February 1985) Abstract--1. Initial velocity and product inhibition studies of Mn:+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. 2. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. 3. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzymePEP-ATP.
INTRODUCTION
notation are according to Cleland (1970). Because of the uncertainty that exists as to the nature of the true substrates for pyruvate kinase, total chemical concentrations of reactants are considered. Phosphoenolpyruvate (PEP), tetrasodium fructose-l,6bisphosphate (FDP), disodium ADP, disodium ATP, NADH, lactate dehydrogenase, pyruvate, MnC12, MgSO4 and trizma base were purchased from Sigma Chemical Co. All other chemicals were analytical grade.
Previous studies from this laboratory described the purification and some properties of a Type-M2 pyruvate kinase from the muscle of the sea mollusc Concholepas concholepas (Le6n et al., 1982; Morfin et al., 1983; Gonzfilez et al., 1984). In the presence of M g 2÷, the enzyme is allosterically activated by fructose-l,6-bisphosphate and the activation is accompanied by the loss of cooperative response to the substrate phosphoenolpyruvate and the loss of sensitivity to the negative effectors alanine and phenylalanine. These effects are also observed in the absence of fructose-l,6-bisphosphate when M g 2÷ is replaced by M n 2÷ as the divalent metal activator of the enzyme. In view of these similarities, we examined in more detail the kinetic properties of the noncooperative species of pyruvate kinase from C. con-
RESULTS AND DISCUSSION
Initial velocity studies Steady state kinetic studies were carried out by using various concentrations of one substrate in the presence of different fixed concentrations of the second substrate. With both the Mn2+-activated enzyme and the M g 2+-enzyme in the presence of F D P , both pairs of substrates gave intersecting lines in Lineweaver-Burk plots. Moreover, all replots for slopes and intercepts were linear. In the range of concentrations used in the present studies, substrate inhibition by A D P was not apparent. Confirming previous reports (Le6n et al., 1982; Gonzfilez et al., 1984), substrate inhibition by A D P was observed only at concentrations higher than 1.3-2 raM. The initial velocity data indicated that both enzyme species follow a sequential mechanism where a central ternary complex must form before the release of any product. Examples of typical results are shown in Fig. l and Table 1 shows the kinetic constants determined from secondary plots of slopes and intercepts vs reciprocal concentrations of the changing fixed substrate.
cholepas. MATERIALS AND M E T H O D S
Pyruvate kinase from the muscle of C. concholepas was purified as already described (Le6n et al., 1982). The metal ions were added to obtain an excess of 4 mM over the total initial concentrations of phosphoenolpyruvate (PEP), ADP adn ATP and fructose-l,6-bisphosphate (FDP) was used at a concentration of 0.1 mM. In initial velocity and ATP-inhibition studies, lactate dehydrogenase was used as a coupling enzyme (Bucher and Pfleiderer, 1955). Pyruvate-inhibition studies were performed by using the direct spectrophotometric assay of Pon and Bondar (1967). All experiments were performed at 25°C in the presence of 0.1 M KCI and 0.1 M Tris-HCl was used as a buffer of pH 7.4. Reactions were initiated by adding the enzyme pyruvate kinase to the substrate buffer solution previously equilibrated at 25°C. Initial velocities were determined from the slopes of the recorded lines. Reciprocal velocities were plotted graphically against reciprocals of substrate concentrations and the results were analyzed by linear regression by using the method of least squares. The kinetic constants were determined from intercept and slope replots of the double reciprocal plots. Nomenclature and
Product inhibition studies T o examine in more detail the interaction of reactants with the enzymes, product inhibition studies were performed. With both enzymatic species, A T P behaved as a non-competitive inhibitor with respect to both A D P and PEP. Significant differences were 63
64
NELSON CARVAJAL et
al.
ADP,mM 4
A
PEP,rnM
5
B
/'
.o.o8
//I" 0.02
i
5
10
15 20 ( I//ADP)m M-I
o08
0
2.5
75
12.5 ( I / p E P)rnM -I
Fig. 1. Initial velocity studies of the reaction catalyzed by pyruvate kinase. (A) Mn2+-activated enzyme and (B) FDP-modified Mg2÷-activated enzyme. The initial rate measured (v) is expressed as a ratio of the rate (Vr) measured in the presence of 0.2 mM ADP and 0.4mM PEP for the Mn2÷-enzyme and 0.2mM ADP, 0.4 PEP and 0.1 mM FDP for the Mg2+-enzyme.
observed, however, in the inhibition caused by pyruvate. In fact, whereas with the Mn2+-enzyme pyruvate was a competitive inhibitor with respect to ADP and a non-competitive inhibitor with respect to PEP, with the FDP-modified Mg2÷-activated enzyme both inhibitions by pyruvate were competitive. All inhibitions examined in this study were linear and constants calculated from secondary plots of slopes and intercepts are shown in Tables 2 and 3. The results obtained favour an ordered sequential mechanism for the Mn2+-activated enzyme and a random combination of reactants with the Mg 2+enzyme in the presence of FDP. The discrepancy between the predicted competitive inhibition and the observed non-competitive pattern of ATP inhibition on the Mg2+-activated species would be explained by mixed product and dead-end inhibition caused by ATP. In fact, the formation of the enzyme-ADP-ATP and enzyme-PEP-ATP dead-end complexes is in accord with our results. Saturation with any of the
Table 1. Kinetic constants from initial velocity studies E-Mn
E-FDP-Mg 2+ (raM)
0.07 0.04 0.55
0,05 0,02 0.45
Constant Ka (ADP) Kb (PEP) Kia
Table 2. Kinetic constants from product inhibition studies of Mn2+-activated pyruvate kinase Inhibitor
Variable substrate
Pattern*
Kis
ATP ATP Pyruvate Pyruvate
ADP PEP ADP PEP
NC NC C NC
Table 3. Kinetic constants from product inhibition studies of FDP-modified Mg2+-activated pyruvate kinase Inhibitor
Variable substrate
Pattern*
ATP ATP Pyruvate Pyruvate
ADP PEP ADP PEP
NC NC C C
Kii? (mM)
1.9 0.5 3.6 3.0
substrates cannot prevent ATP from combining to form the corresponding dead-end complex and the resulting intercept effect explains the non-competitive character of the inhibition. The linearity of ATP inhibitions argue against the formation of an enzymeATP-ATP dead-end complex, at least in the range of concentrations used in our studies (up to 12mM). The existence of two types of binding sites for ADP has been suggested by kinetic protection studies of rabbit muscle pyruvate kinase (Mildvan and Cohn, 1966) and by X-ray crystallographic analysis of the cat muscle enzyme (Stammers and Muirhead, 1975). On the other hand, several reports has been concerned with substrate inhibitions of pyruvate kinase and the apparent ease with which the enzyme forms dead-end complexes (Janson and Cleland, 1974; Giles et al., 1976a; Newton et al., 1976; Giles and Poat, 1980; Munday et al., 1980). In one of these reports, Giles and Poat (1980) described that in the presence of FDP, the enzymes from Carcinus maenas forms five dead-end complexes and that pyruvate is the only reactant not exhibiting substrate inhibition. Apparently, therefore, for substrate inhibition to occur with this enzyme, the presence of a phosphate group is required. With pyruvate kinase from C. concholepas we never observed inhibition by excess PEP and only the nucleotide reactants seems to have tendency to interact with the enzyme in a dead-end combination.
2.1 4.1 -12.6
*C, competitive; NC, non-competitive. tApparent inhibition constants were calculated from replots of slopes (Kis) and intercepts (Kii).
Kis
Kilt (mM)
8.8 3.2 2.3 3.0
3.9 6.7 ---
*C, competitive; NC, non-competitive. tApparent inhibition constants were calculated from replots of slopes (Kis) and intercepts (Kii).
PK activated by FDP This is expressed by substrate inhibition by A D P of both the Mn2+-activated and the Mg2+-activated enzymes and the mixed product and dead-end inhibition caused by A T P on the Mg2+-activated species. The suggestion that pyruvate kinase from C. concholepas catalyzes a sequential mechanism is in agreement with the results of steady state kinetic studies of the enzyme from various sources (Macfarlane and Ainsworth, 1972; D a n n and Britton, 1977; Reynard et al., 1961; Ainsworth and Macfarlane, 1973; Giles and Poat, 1980). One exception would be the suggestion of a ping-pong mechanism for the enzyme from pig liver (Macfarlane and Ainsworth, 1974) even though isotope exchange studies indicate that the enzyme catalyzes a sequential mechanism (Giles et al., 1976b). Interestingly, whereas in the presence of M g 2+ and F D P there is a random combination of reactants with pyruvate kinase from C. concholepas, in the presence of M n 2÷ there is a compulsory order of binding so that no binding site exists on the enzyme for PEP until A D P is bound. Acknowledgements--This work was supported by Grant 20.31.09 of the Direcci6n de Investigaci6n, Universidad de Concepci6n. REFERENCES
Ainsworth S. and Macfarlane N. (1973) A kinetic study of rabbit muscle pyruvate kinase. Biochem. J. 131, 223-236. Bucher T. and Pfleiderer G. (1955) Methods in Enzymology, (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 435--440. Academic Press, New York. Cleland W. W. (1970) The Enzymes (Edited by Boyer P. D.), 3rd Edn, Vol. 2, pp. 1-65. Academic Press, New York. Dann L. G. and Britton H. G. (1977) Mechanism of L-pyruvate kinase from rabbit liver. Evidence against phospho-enzyme formation. Biochem. J. 161, 445--448. Giles I. G. and Poat P. C. (1980) A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas. Biochem. J. 185, 289-299. Giles I. G., Poat P. C. and Munday K. A. (1976a) The kinetics of rabbit muscle pyruvate kinase. Initial velocity,
C.B.P. 82/11~E
65
substrate and product inhibition and isotopic exchange studies of the reverse reaction. Biochem. J. 157, 577-589. Giles I. G., Poat P. C. and Munday K. A. (1976b) Isotopic exchange studies on the type-L pyruvate kinase from pig liver. Biochem. Soc. Trans. 4, 1156--1158. GonzAlez R., Carvajal N. and MorAn A. (1984) Differences between magnesium-activated and manganase-activated pyruvate kinase from the muscle of Concholepas concholepas. Comp. Biochem. Physiol. 7813, 389-392. Janson C. A. and Cleland W. W. (1974) The inhibition of acetate, pyruvate and 3-phospboglycerate kinases by Cr ATP. J. biol. Chem. 249, 2567-2571. Le6n O., MorAn A. and GonzAlez R. (1982) Purification and characterization of pyruvate kinase from muscle of the sea mollusc Concholepas concholepas. Comp. Biochem. Physiol. 72B, 65-69. Macfarlane N. and Ainsworth S. (1972) A kinetic study of Baker's yeast pyruvate kinase activated by fructose-l,6disphosphate. Biochem. J. 129, 1035-1047. Macfarlane N. and Ainsworth S. (1974) A kinetic study of pig liver pyruvate kinase activated by fructose1,6,diphosphate. Biochem. J. 139, 499-508. Mildvan A. S. and Cohn M. (1966) Kinetic and magnetic resonance studies of the pyruvate kinase reaction. II Complexes of enzyme, metal and substrates. J. biol. Chem. 241, 1178-1193. MorAn A., GonzAlez R. and Le6n O. (1983) Studies of the effects of phenylalanine and pH on pyruvate kinase from the muscle of the sea mollusc Concholepas concholepas. Comp. Biochem. Physiol. 7511, 603-606. Munday K. A., Giles I. G. and Poat P. C. (1980) Review of the comparative biochemistry of pyruvate kinase. Comp. Biochem. Physiol. 6711, 403--411. Newton C. J., Poat P. C. and Munday K. A. (1976) Substrate inhibition of pyruvate kinase isolated from the leg muscle of Carcinus maenas. Biochem. Soc. Trans. 4, 1158-1160. Pon N. G. and Bondar R. J. L. (1967) A direct spectrophotometric assay for pyruvate kinase. Analyt. Biochem. 19, 272-279. Reynard A., Hass L. F. Jacobsen D. D. and Boyer P. D. (1961) The correlation of reaction kinetics and substrate binding with the mechanism of pyruvate kinase. J. biol. Chem. 236, 2277-2283. Stammers D. K. and Muirhead H. (1975) Threedimensional structure of cat muscle pyruvate kinase at 6 A ° resolution. J. molec. Biol. 95, 213-225.
0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd
Printed in Great Britain
COMPARATIVE KINETIC STUDIES OF Mn 2+-ACTIVATED AND FRUCTOSE- 1,6-P-MODIFIED
Mg2÷-ACTIVATED PYRUVATE KINASE FROM CONCHOLEPAS CONCHOLEPAS NELSON CARVAJAL, RUBY GONZALEZ, ARSENIO MORAN and ANA MARIA OYARCE Departamento de Biologia Molecular, Facultad de Ciencias Biol6gicas y de Recursos Naturales, Universidad de Concepci6n, Concepci6n, Chile
(Received 11 February 1985) Abstract--1. Initial velocity and product inhibition studies of Mn:+-activated and FDP-modified Mg2+-activated pyruvate kinase from Concholepas concholepas, were performed. 2. Evidence is presented to show that the Mn2+-enzyme catalyzes an ordered sequential mechanism, with ADP being the first substrate and pyruvate the last product. 3. The results presented are consistent with a random combination of reactants with the FDP-modified Mg2+-activated enzyme and the formation of the dead-end complexes enzyme ADP-ATP and enzymePEP-ATP.
INTRODUCTION
notation are according to Cleland (1970). Because of the uncertainty that exists as to the nature of the true substrates for pyruvate kinase, total chemical concentrations of reactants are considered. Phosphoenolpyruvate (PEP), tetrasodium fructose-l,6bisphosphate (FDP), disodium ADP, disodium ATP, NADH, lactate dehydrogenase, pyruvate, MnC12, MgSO4 and trizma base were purchased from Sigma Chemical Co. All other chemicals were analytical grade.
Previous studies from this laboratory described the purification and some properties of a Type-M2 pyruvate kinase from the muscle of the sea mollusc Concholepas concholepas (Le6n et al., 1982; Morfin et al., 1983; Gonzfilez et al., 1984). In the presence of M g 2÷, the enzyme is allosterically activated by fructose-l,6-bisphosphate and the activation is accompanied by the loss of cooperative response to the substrate phosphoenolpyruvate and the loss of sensitivity to the negative effectors alanine and phenylalanine. These effects are also observed in the absence of fructose-l,6-bisphosphate when M g 2÷ is replaced by M n 2÷ as the divalent metal activator of the enzyme. In view of these similarities, we examined in more detail the kinetic properties of the noncooperative species of pyruvate kinase from C. con-
RESULTS AND DISCUSSION
Initial velocity studies Steady state kinetic studies were carried out by using various concentrations of one substrate in the presence of different fixed concentrations of the second substrate. With both the Mn2+-activated enzyme and the M g 2+-enzyme in the presence of F D P , both pairs of substrates gave intersecting lines in Lineweaver-Burk plots. Moreover, all replots for slopes and intercepts were linear. In the range of concentrations used in the present studies, substrate inhibition by A D P was not apparent. Confirming previous reports (Le6n et al., 1982; Gonzfilez et al., 1984), substrate inhibition by A D P was observed only at concentrations higher than 1.3-2 raM. The initial velocity data indicated that both enzyme species follow a sequential mechanism where a central ternary complex must form before the release of any product. Examples of typical results are shown in Fig. l and Table 1 shows the kinetic constants determined from secondary plots of slopes and intercepts vs reciprocal concentrations of the changing fixed substrate.
cholepas. MATERIALS AND M E T H O D S
Pyruvate kinase from the muscle of C. concholepas was purified as already described (Le6n et al., 1982). The metal ions were added to obtain an excess of 4 mM over the total initial concentrations of phosphoenolpyruvate (PEP), ADP adn ATP and fructose-l,6-bisphosphate (FDP) was used at a concentration of 0.1 mM. In initial velocity and ATP-inhibition studies, lactate dehydrogenase was used as a coupling enzyme (Bucher and Pfleiderer, 1955). Pyruvate-inhibition studies were performed by using the direct spectrophotometric assay of Pon and Bondar (1967). All experiments were performed at 25°C in the presence of 0.1 M KCI and 0.1 M Tris-HCl was used as a buffer of pH 7.4. Reactions were initiated by adding the enzyme pyruvate kinase to the substrate buffer solution previously equilibrated at 25°C. Initial velocities were determined from the slopes of the recorded lines. Reciprocal velocities were plotted graphically against reciprocals of substrate concentrations and the results were analyzed by linear regression by using the method of least squares. The kinetic constants were determined from intercept and slope replots of the double reciprocal plots. Nomenclature and
Product inhibition studies T o examine in more detail the interaction of reactants with the enzymes, product inhibition studies were performed. With both enzymatic species, A T P behaved as a non-competitive inhibitor with respect to both A D P and PEP. Significant differences were 63
64
NELSON CARVAJAL et
al.
ADP,mM 4
A
PEP,rnM
5
B
/'
.o.o8
//I" 0.02
i
5
10
15 20 ( I//ADP)m M-I
o08
0
2.5
75
12.5 ( I / p E P)rnM -I
Fig. 1. Initial velocity studies of the reaction catalyzed by pyruvate kinase. (A) Mn2+-activated enzyme and (B) FDP-modified Mg2÷-activated enzyme. The initial rate measured (v) is expressed as a ratio of the rate (Vr) measured in the presence of 0.2 mM ADP and 0.4mM PEP for the Mn2÷-enzyme and 0.2mM ADP, 0.4 PEP and 0.1 mM FDP for the Mg2+-enzyme.
observed, however, in the inhibition caused by pyruvate. In fact, whereas with the Mn2+-enzyme pyruvate was a competitive inhibitor with respect to ADP and a non-competitive inhibitor with respect to PEP, with the FDP-modified Mg2÷-activated enzyme both inhibitions by pyruvate were competitive. All inhibitions examined in this study were linear and constants calculated from secondary plots of slopes and intercepts are shown in Tables 2 and 3. The results obtained favour an ordered sequential mechanism for the Mn2+-activated enzyme and a random combination of reactants with the Mg 2+enzyme in the presence of FDP. The discrepancy between the predicted competitive inhibition and the observed non-competitive pattern of ATP inhibition on the Mg2+-activated species would be explained by mixed product and dead-end inhibition caused by ATP. In fact, the formation of the enzyme-ADP-ATP and enzyme-PEP-ATP dead-end complexes is in accord with our results. Saturation with any of the
Table 1. Kinetic constants from initial velocity studies E-Mn
E-FDP-Mg 2+ (raM)
0.07 0.04 0.55
0,05 0,02 0.45
Constant Ka (ADP) Kb (PEP) Kia
Table 2. Kinetic constants from product inhibition studies of Mn2+-activated pyruvate kinase Inhibitor
Variable substrate
Pattern*
Kis
ATP ATP Pyruvate Pyruvate
ADP PEP ADP PEP
NC NC C NC
Table 3. Kinetic constants from product inhibition studies of FDP-modified Mg2+-activated pyruvate kinase Inhibitor
Variable substrate
Pattern*
ATP ATP Pyruvate Pyruvate
ADP PEP ADP PEP
NC NC C C
Kii? (mM)
1.9 0.5 3.6 3.0
substrates cannot prevent ATP from combining to form the corresponding dead-end complex and the resulting intercept effect explains the non-competitive character of the inhibition. The linearity of ATP inhibitions argue against the formation of an enzymeATP-ATP dead-end complex, at least in the range of concentrations used in our studies (up to 12mM). The existence of two types of binding sites for ADP has been suggested by kinetic protection studies of rabbit muscle pyruvate kinase (Mildvan and Cohn, 1966) and by X-ray crystallographic analysis of the cat muscle enzyme (Stammers and Muirhead, 1975). On the other hand, several reports has been concerned with substrate inhibitions of pyruvate kinase and the apparent ease with which the enzyme forms dead-end complexes (Janson and Cleland, 1974; Giles et al., 1976a; Newton et al., 1976; Giles and Poat, 1980; Munday et al., 1980). In one of these reports, Giles and Poat (1980) described that in the presence of FDP, the enzymes from Carcinus maenas forms five dead-end complexes and that pyruvate is the only reactant not exhibiting substrate inhibition. Apparently, therefore, for substrate inhibition to occur with this enzyme, the presence of a phosphate group is required. With pyruvate kinase from C. concholepas we never observed inhibition by excess PEP and only the nucleotide reactants seems to have tendency to interact with the enzyme in a dead-end combination.
2.1 4.1 -12.6
*C, competitive; NC, non-competitive. tApparent inhibition constants were calculated from replots of slopes (Kis) and intercepts (Kii).
Kis
Kilt (mM)
8.8 3.2 2.3 3.0
3.9 6.7 ---
*C, competitive; NC, non-competitive. tApparent inhibition constants were calculated from replots of slopes (Kis) and intercepts (Kii).
PK activated by FDP This is expressed by substrate inhibition by A D P of both the Mn2+-activated and the Mg2+-activated enzymes and the mixed product and dead-end inhibition caused by A T P on the Mg2+-activated species. The suggestion that pyruvate kinase from C. concholepas catalyzes a sequential mechanism is in agreement with the results of steady state kinetic studies of the enzyme from various sources (Macfarlane and Ainsworth, 1972; D a n n and Britton, 1977; Reynard et al., 1961; Ainsworth and Macfarlane, 1973; Giles and Poat, 1980). One exception would be the suggestion of a ping-pong mechanism for the enzyme from pig liver (Macfarlane and Ainsworth, 1974) even though isotope exchange studies indicate that the enzyme catalyzes a sequential mechanism (Giles et al., 1976b). Interestingly, whereas in the presence of M g 2+ and F D P there is a random combination of reactants with pyruvate kinase from C. concholepas, in the presence of M n 2÷ there is a compulsory order of binding so that no binding site exists on the enzyme for PEP until A D P is bound. Acknowledgements--This work was supported by Grant 20.31.09 of the Direcci6n de Investigaci6n, Universidad de Concepci6n. REFERENCES
Ainsworth S. and Macfarlane N. (1973) A kinetic study of rabbit muscle pyruvate kinase. Biochem. J. 131, 223-236. Bucher T. and Pfleiderer G. (1955) Methods in Enzymology, (Edited by Colowick S. P. and Kaplan N. O.), Vol. 1, pp. 435--440. Academic Press, New York. Cleland W. W. (1970) The Enzymes (Edited by Boyer P. D.), 3rd Edn, Vol. 2, pp. 1-65. Academic Press, New York. Dann L. G. and Britton H. G. (1977) Mechanism of L-pyruvate kinase from rabbit liver. Evidence against phospho-enzyme formation. Biochem. J. 161, 445--448. Giles I. G. and Poat P. C. (1980) A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas. Biochem. J. 185, 289-299. Giles I. G., Poat P. C. and Munday K. A. (1976a) The kinetics of rabbit muscle pyruvate kinase. Initial velocity,
C.B.P. 82/11~E
65
substrate and product inhibition and isotopic exchange studies of the reverse reaction. Biochem. J. 157, 577-589. Giles I. G., Poat P. C. and Munday K. A. (1976b) Isotopic exchange studies on the type-L pyruvate kinase from pig liver. Biochem. Soc. Trans. 4, 1156--1158. GonzAlez R., Carvajal N. and MorAn A. (1984) Differences between magnesium-activated and manganase-activated pyruvate kinase from the muscle of Concholepas concholepas. Comp. Biochem. Physiol. 7813, 389-392. Janson C. A. and Cleland W. W. (1974) The inhibition of acetate, pyruvate and 3-phospboglycerate kinases by Cr ATP. J. biol. Chem. 249, 2567-2571. Le6n O., MorAn A. and GonzAlez R. (1982) Purification and characterization of pyruvate kinase from muscle of the sea mollusc Concholepas concholepas. Comp. Biochem. Physiol. 72B, 65-69. Macfarlane N. and Ainsworth S. (1972) A kinetic study of Baker's yeast pyruvate kinase activated by fructose-l,6disphosphate. Biochem. J. 129, 1035-1047. Macfarlane N. and Ainsworth S. (1974) A kinetic study of pig liver pyruvate kinase activated by fructose1,6,diphosphate. Biochem. J. 139, 499-508. Mildvan A. S. and Cohn M. (1966) Kinetic and magnetic resonance studies of the pyruvate kinase reaction. II Complexes of enzyme, metal and substrates. J. biol. Chem. 241, 1178-1193. MorAn A., GonzAlez R. and Le6n O. (1983) Studies of the effects of phenylalanine and pH on pyruvate kinase from the muscle of the sea mollusc Concholepas concholepas. Comp. Biochem. Physiol. 7511, 603-606. Munday K. A., Giles I. G. and Poat P. C. (1980) Review of the comparative biochemistry of pyruvate kinase. Comp. Biochem. Physiol. 6711, 403--411. Newton C. J., Poat P. C. and Munday K. A. (1976) Substrate inhibition of pyruvate kinase isolated from the leg muscle of Carcinus maenas. Biochem. Soc. Trans. 4, 1158-1160. Pon N. G. and Bondar R. J. L. (1967) A direct spectrophotometric assay for pyruvate kinase. Analyt. Biochem. 19, 272-279. Reynard A., Hass L. F. Jacobsen D. D. and Boyer P. D. (1961) The correlation of reaction kinetics and substrate binding with the mechanism of pyruvate kinase. J. biol. Chem. 236, 2277-2283. Stammers D. K. and Muirhead H. (1975) Threedimensional structure of cat muscle pyruvate kinase at 6 A ° resolution. J. molec. Biol. 95, 213-225.