Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 270, No. 2, May 1, pp. 647-654,1989

Steady-State

Kinetic Studies of the Metal Ion-Dependent Decarboxylation of Oxalacetate Catalyzed by Pyruvate Kinase’ DENNIS

M. KIICK’

AND

WM. WALLACE

CLELAND3

Department of Biochemistry, University of Wisconsin, Ma.dism, Wimmsin 5$706 Received October 25.1988, and in revised form January

9,1989

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co’+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the Vfor oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the PKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mgz+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity. D 1989 Academic Press, Inc.

ben and Cohn (3) have shown that the enzyme, in the absence of substrates, binds one mole of divalent cation per mole of subunit. This cation has been postulated to act as a bridge to help bind the phosphate group of phosphoenolpyruvate (4), and Lo-

For pyruvate kinase, three different metal ions are required for the phosphoryl transfer from phosphoenolpyruvate to adenosine diphosphate: M$+, K+, and the MgC in the Mg-ADP* complex (1,2). Reu1 This research was supported by a National Institutes of Health postdoctoral fellowship (GM10857) to D.M.K. and a grant from the National Institutes of Health (GM18938) to W. W. Cleland. ’ Present address: Department of Biochemistry, University of Tennessee, Memphis, College of Medicine, 866 Madison Avenue, Memphis, TN 38163. a To whom correspondence should be addressed. ’ Abbreviations used: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Ches, 2-(N-cyclohexylamino)ethanesulfonic acid; DEA, diethanolamine;

DTNB, 5,5’-dithiobis(2-nitrobenzoate); DTT, dithiothrietol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; LDH, lactate dehydrogenase; Mes, 2-(N-morpholino)ethanesulfonic acid; NADH, nicotinamide adenine dinucleotide; OAA, oxalacetic acid; PEP, phosphoenolpyruvate; Pipes, piperazine-N,N’bis(2-ethanesulfonic acid); Taps, 3Qtris(hydroxymethyl)methyl]amino]propanesulfonic acid; TBA, tributylammonium; TEA, triethanolamine; TNB, thionitrobenzoate. 647

6663-9861/89 $3.66 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

648

KIICK AND CLELAND

dato and Reed (5) have shown that ATP acts as a bridging ligand between the two divalent metal ions on enzyme. Thus, the metal ion promotes the phospho transfer between the anionic substrates. The role of the monovalent cation is to elicit a conformational change from the enzyme that is essential for formation of the catalytically active enzyme-substrate complex (6). Pyruvate kinase will also catalyze the metal ion-dependent decarboxylation of oxalacetate (7-9) and this reaction has been shown to be catalyzed in the same site on the enzyme as the phospho transfer reaction (10). Dougherty and Cleland (11) have studied the kinetics of phosphoryl transfer and oxalacetate decarboxylation, as well as the kinetics of some alternate reactions catalyzed by pyruvate kinase in the presence of Mg2f (12). Results of the above work indicate that three residues with pK values of 7.0,8.3, and 9.2 are important for catalysis and binding of substrates. In conjunction with X-ray crystallographic data (13), Dougherty and Cleland (11, 12) assigned one of the pK values (7.0) to an enzymatic glutamate residue which binds the free divalent cation. The second pK value (8.3) was assigned to an enzymatic lysine residue which presumably donates a proton to the enolate of pyruvate upon phosphoryl transfer to form ATP and pyruvate, and the third pKvalue (9.2) was postulated to be that of metal ion bound water. The present studies were undertaken to explore the role of the divalent metal in the OAA decarboxylation reaction and how it possibly relates to the phosphoryl transfer reaction catalyzed by pyruvate kinase. As has been shown by Dougherty and Cleland (ll), the decarboxylation reaction is divalent metal ion dependent, and as will be shown in this study it is nucleotide independent and also monovalent metal ion independent. Thus, studying the chemistry of the divalent cation should allow one to draw unambiguous conclusions concerning the overall role of the divalent cation in this reaction. EXPERIMENTAL

PROCEDURES

Ch.ew&als and enzymes. Pyruvate kinase was obtained from Sigma Chemical Co. as a lyophilized pow-

der or in 50% glycerol, 10 mM phosphate, pH 7.0. The LDH used in the coupled assays was also purchased as a lyophilized powder from Sigma (rabbit muscle) or as a solution in 50% glycerol, pH 6.5, from Boehringer-Mannheim (hog muscle). Both enzymes were used without further purification. The ADP-dependent conversion of PEP to pyruvate was monitored spectrophotometrically at 340 nm by means of a LDH-coupled assay. Pyruvate kinase specific activity was calculated using a subunit molecular weight of 59,250(3,22), and an enzyme active site concentration was calculated using an extinction coefficient at 236 nm for a 1% solution of 5.4 cm-’ (23). The calculated phosphorylation activity of the kinase was in reasonable agreement with previous literature values (3, 22). NADH and DTT were purchased from Boehringer-Mannheim. Good’s buffers were from Research Organics. All solutions were made with double distilled, demineralized water and were stored at 4°C in polyethylene bottles. Initial velocity studies. Pyruvate kinase-catalyzed decarboxylation of oxalacetate was assayed spectrophotometrically with a Beckman DU monochromator and a Gilford OD converter, using a Leeds-Northrop chart recorder with multispeed drive. All assays were carried out at 25°C and reaction rates were measured by coupling the pyruvate produced to the oxidation of NADH in the presence of excess LDH (14). The temperature was maintained with a circulating water bath with the capacity to heat and cool the thermospacers of the Beckman DU. Reaction cuvettes were 1 cm in path length and 1 or 3 ml in volume. All cuvettes were incubated for at least 5 min in the water bath prior to insertion into the cell compartment and initiation of the reaction. Assay temperatures were routinely monitored with a YSI tele-thermometer while the cuvette was still in the cell compartment. A typical assay contained 0.1 M buffer (see pH studies below), 0.2 mM NADH, 36 U/ml LDH, and variable concentrations of divalent metal and OAA (correcting for the amount of metal-chelate complex formed (15)). The reaction was initiated by the addition of pyruvate kinase (0.15 mg/ml) and the disappearance of NADH was monitored at 340 nm, &,, = 6.22 mM-’ cm-‘. Initial velocity data obtained in the presence of PEP, the metal nucleotide complexes (Cr-ATP, MnATP, or Mn-ADP), or oxalate were collected in a manner similar to that outlined above except the inhibitor concentrations were corrected for metal-chelate complex formation using the appropriate K, values (15). Velocity as a function of enzyme concentration was determined at pH 6.0, 8.5, and 9.5 when Co*+, Mgz+, or Mn*+ was used as the divalent cation and also at the highest inhibitor concentrations used in obtaining the inhibition patterns. Enzymatic rates were corrected for the amount of nonenzymatic metal iondependent decarboxylation that took place in the re-

OXALACETATE

DECARBOXYLATION

action cuvettes. In all cases the correction was less than 10% of the overall enzymatic rate. pHstudia. Determination of the divalent metal ion Ki values and the Vand V/K for OAA were carried out by varying the levels of OAA at the desired fixed levels of the appropriate metal ion and obtaining the initial velocity. The Ki for inhibitors vs either OAA or metal ion were determined by varying the concentrations of substrate at several different inhibitor concentrations (including zero). All assays reflected initial velocity conditions with less than 10% of the limiting reactant utilized over the time course of the reaction. The pH ranges of the buffers were: Mes, 5.56.5; Pipes, 6.5-7.5; Hepes, 7.0-8.0; Taps, 8.0-9.0; TEA, 8.0-8.5; DEA, 8.5-9.5; Ches, 9.0-10.0; ethanolamine, 9.5-10.0. Unless otherwise noted, all buffers were titrated to the appropriate pH with KOH. In all cases, sufficient overlaps were obtained when buffers were changed so that correction could be made for spurious buffer effects5 (16). The pH of the reaction mixture was measured with a Beckman Psi 21 pH meter with a combined microelectrode before and after sufficient data were collected for determination of initial velocities. Negligible pH changes were observed before and after reaction. Data analysis. Reciprocal initial velocities were plotted as a function of reciprocal substrate concentrations. Data were analyzed using the appropriate rate equations and whenever possible by using the FORTRAN progams of Cleland (17). The points in the figures are the experimentally determined values, while the curves are calculated from fits of the data using the appropriate equation. Initial velocity data conforming to rapid equilibrium ordered addition of the metal prior to oxalacetate were fitted to VAB “=K,K,+K,A+AB’

BY PYRUVATE

649

KINASE

VA ’ = K, + A(1 + I/K,)

141

’

In the above equations, A and B are the reactant concentrations and I is the inhibitor concentration; K,, and Kb are the Michaelis constants. The K, is the slope inhibition constant, and the Kii is the intercept inhibition constant. Data for pH profiles which decreased with a slope of 1 or 2 at high pH were fitted to Eqs. [5] and [6], respectively: log Y = log kH log Y = log

1

C 1-k K,/H + K,K2/H2 ’

Fl

Data for the pKi profiles in which the pKi decreased at low pH and increased at high pH from a constant value were fitted to lwY=lw

C(1 + K,/H) l+H,K 1

.

In Eqs. [5], [6], and [7], Kl and K2 represent the dissociation constants for enzyme groups, Y is the value of the parameter observed as a function of pH, and C is the pH-independent value of X In all cases, the best fit of the data was chosen on the basis of the lowest values of the standard errors of the fitted parameters and the lowest value of c. c is defined as the sum of the squares of the residuals divided by the degrees of freedom, where degrees of freedom is equal to the number of points minus the number of parameters (17). RESULTS AND DISCUSSION

Initial velocity studies in the absence of inhibitors. The decarboxylation of OAA by pyruvate kinase displays rapid equilibrium ordered kinetics with the divalent metal (either Co2+,as shown in Fig. 1, or VA Mn2+, as shown in Fig. 2) adding to enzyme ’ = K.(l + I/K,) + A prior to OAA. Dougherty and Cleland (11) have also observed a similar pattern for VA 131 OAA decarboxylation with M$+. Thus, the ’ = K,,(l + I/K,) + A(1 + I/Kii) kinetic mechanism for OAA decarboxylation appears to be the same regardless of 5 Small amine cations (monmethylammonium or the type of divalent cation. Also, from the dimethylammonium) have been shown to bind to py- pH dependence of these kinetic data (see ruvate kinase (6). However, in this study, data ac- pH studies, below) the kinetic mechanism quired in the presence of ethanolamine, the smallest has been determined to be pH indepenof the cationic acid buffers used, as well as DEA and TEA, when compared to equivalent data acquired in dent. However, above pH 8 in the presence of Mn2+, while the double-reciprocal plots the presence of Ches and/or Taps, relatively large still appear to intersect on the ordinate cations which have been shown not to bind to pyruwhen l/v is plotted vs l/[OAA], there is exvate kinase (6), were the same in all cases.

Data for linear competitive, noncompetitive, and uncompetitive inhibition was fitted using Eqs. [2], [3], and [4], respectively:

KIICK

AND

co** (mM) pu

7.4 ./.05

60.

10

40 IIOAA

70

.

100 (mM-11

FIG. 1. Pyruvate kinase initial velocity pattern for OAA decarboxylation with Co’+. The data were obtained in the presence of 0.1 M TBA-Hepes, pH 7.4. The open circles represent data obtained in the presence of 0.1 M KCl. The lines drawn are from a fit of the data to Eq. [l].

treme curvature, indicating apparent positive cooperativity with respect to OAA. Below pH 8 all double-reciprocal plots are linear. Monovalent cation eflecta Initial velocity patterns obtained in the absence and presence of 100 mM KC1 also display rapid equilibrium ordered kinetics and have the same kinetic parameters (Fig. 1). Nowak (6) has shown that alternate amine cations can bind at the Kf binding site and cause a conformational change in the enzyme which is necessary for the phosphoryl transfer substrates to form a catalytically active compex. However, very large monovalent cations, as is the case with TBA, will not bind and elicit the conformation change in the enzyme that is necessary for the phosphoryl transfer substrates to bind and react. Thus, from the inhibitor studies (see below) carried out in the presence and absence of monovalent cations (TBA was the counterion for all substrates and buffers) and from the initial velocity data obtained in the absence and presence of K+, we conclude that K+ is not required for the enzyme-catalyzed decarboxylation of OAA. Moreover, the conformational change ellicited by K+, which is required for proper binding and catalysis of substrates of the phosphoryl transfer reaction (5), is not re-

CLELAND

quired for pyruvate kinase to decarboxylate OAA. This implies that although both reactions are catalyzed in the same site on the enzyme, the tertiary enzyme structure, and thus the overall geometry of the substrates relative to the enzyme residues participating in the chemistry in the site, most likely is different. This agrees with the chemical inactivation data of Jursinic and Robinson (10) who suggested different amino acid residues in the active site participate in the two reactions. Inhibition studies. A summary of the inhibition data is shown in Table I. As shown, PEP is a competitive inhibitor of OAA decarboxylation, and exhibits uncompetitive inhibition when the metal is the variable substrate. PEP, one of the phosphoryl transfer substrates for pyruvate kinase, effectively binds as a dead-end inhibitor. Uncompetitive inhibition by a dead-end inhibitor will be observed only if the inhibitor binds to the enzyme after the variable substrate6 is bound. Therefore, knowing that the metal binds to enzyme in an equilibrium ordered fashion prior to OAA allows one to write the following mechanism: metal

E

1

products

OAA

EM

1 (

>

EMP

EM

1k EM-PEP These data indicate that PEP binds either very synergistically or only to the enzymemetal complex and not to free enzyme. Note that the && and & values from the inhibition patterns for PEP are essentially equal and agree with the PEP Ki data of Jursinic and Robinson (lo), further suggesting that PEP binds only to the enzyme-metal complex. Thus, although the 6 Although the metal ion is not tranformed during the course of the reaction, the rate is directly dependent on the uncomplexed divalent metal ion concentration and therefore the metal ion can be considered a pseudoreactant.

OXALACETATE

DECARBOXYLATION

BY PYRUVATE

651

KINASE

TABLE I INHIBITION CONSTANTSFORPYRUVATEKINASE~ Inhibitor

Variable substrate

Fixed substrate

Type inhibition*

KS

&

(FM)

(PM)

Cr-ATP

OAA

C

74 + 6

Mn-ATP

OAA

Mn-ADP

OAA

PEP

OAA

PEP

Mn2+

Mn2+ W1rM Mn2+ 25 PM Mn2+ 25cm Mn2+ WPM OAA 75 pM

C NC C UC

102 f

8

50 *lo

266 +50

2.2+ 0.1 3.3 + 0.1

“Determined in the presence of 0.1 M TBA-Hepes, pH 7.3. * C, competitive inhibition. Data fitted to Eq. 121.NC, noncompetitive inhibition. Data fitted to Eq. [3]. UC, uncompetitive inhibition. Data fitted to Eq. [4].

kinetic mechanism of phosphoryl transfer is random with respect to addition of MgATP and PEP (18), the metal ion must be bound to the enzyme prior to PEP. Consistent with the above model, direct binding studies by Nowak and Mildvan (4) demonstrated that there was very strong sinergism in the binding of Mn2+ and PEP, since the binding of PEP to free enzyme is much weaker than the binding of Mn2+. The OAA decarboxylation is also inhibited competively by either Cr-ATP or MnATP and noncompetively by Mn-ADP.

Mr? (mh4) pH 120

7.3

/

I

.Ol

i

I . 2

I 0 l/OAA

14

20

(mM-‘1

FIG. 2. Pyruvate kinase initial velocity pattern for OAA decarboxylation with Mn2+.The lines drawn are from a fit of the data to Eq. [l].

This suggests that the y phosphate of ATP may overlap with some portion of OAA when it is bound to the enzyme. pH studies. The PKi values for all three divalent metal ions that have been studied to date (Co2+, Mn2+, and Mgs+), while exhibiting differing absolute values, decrease below a pK of 7 and increase above a pK of 9.0 as shown in Fig. 3. A similar curve was reported for Mn2+ by Mildvan and Cohn (19) on the basis of direct binding studies. Since there is no perturbation of either of these pK values with differing divalent metals (as would be expected if one of the observed pK values corresponded to water coordinated to a metal ion), the pK values must be attributable to enzymatic residues. The low pK value probably reflects a glutamate residue liganded to the metal as has been previously suggested (12). The pK of this residue will not appear in the pH profiles where the metal ion is saturating, since metal ion coordination will prevent protonation. Since metal ions bind more tightly above a pK of 9, this pK will be lower in the enzyme-metal ion complex by the log of the ratio of metal ion dissociation constants below and above this PK. The tighter binding of the metal ion above this pK presumably results from eliminating unfavorable electrostatic interactions between the

652

KIICK AND CLELAND

log

II

I

6

7

6

I

*I

9

10

PH

FIG. 3. The pH dependence of the divalent metal ion dissociation constants for pyruvate kinase. The open circles represent the pKi for Co2+obtained from a fit to Eq. [l] of initial velocity data similar to those shown in Fig. 1. The calculated l/Kivalues were fitted to Eq. [Sl, resulting in the curve shown and two pK values of 7.0 + 0.2 and 8.8 f 0.2. The closed circles represent pKi values for Mn2+ obtained from a fit to Eq. [I] of the initial velocity data below pH 8, similar to those shown in Fig. 2. Above pH 8, due to the observed OAA positive cooperativity, data for Mn2+ saturation curves were fitted to the Michaelis-Menten equation. The uncomplexed [OAA] was maintained at or below 0.05 mre. The calculated l/Ki values were fitted to Eq. [6], resulting in the curve shown and two pK values of 7.0 zt 0.1 and 8.9 -t 0.1. Beneath the Co2+curve is drawn a theoretical curve for Me using two pK values of 7.0 and 9.1 and the pa-independent value of the Ki as reported by Dougherty and Cleland (11). The crosses represent pKi values for MC from initial velocity data obtained under conditions similar to those described by Dougherty and Cleland (11) and fitted to Eq. Ill-

positive charged metal ion and the enzyme residue with pK of 9, if this residue is a cationic acid, or enhanced electrostatic interaction between the metal ion and the anionic form of this residue if it is a neutral acid. This enzyme residue may be the lysine which acts as the acid-base catalyst for enolization or ketonization of pyruvate (13). With Mp, the lysine exhibits a pKof 8.2 in the E-Mg complex (13), corresponding to ea. sixfold tighter binding of Mgz when the lysine is not charged. This lysine pK does not appear in the pH profiles of reactions other than those involving enolization (11,12), and is also not observed in the decarboxylation of oxalacetate.7 This group does affect the detritiar Although the conversion of the enolate of pyruvate to ketopyruvate is part of the reaction, this is

log

V/K

v

I/

-‘-‘\,

]

-1

-2 6

7

6

9

10

PH FIG. 4. The pH dependence of the kinetic parameters for OAA decarboxylation catalyzed by pyruvate kinase in the presence of Co2+.The points represent values calculated from a fit of the data to Eq. [l]. The free [Co2+]was varied from 0.1 to 10 times its Kj value and the uncomplexed [OAA] was varied from 0.1 to 10 times its Km value. The Vvalues were fitted to Eq. [5], resulting in the curve shown and a pK value of 8.0 f 0.1. The V/KoAA decreases below two pK values. The V/K values were fitted using Eq. [Sl, resulting in the curve shown and in an average of the two pK values being 7.8 f 0.1.

tion of pyruvate catalyzed by pyruvate kinase, however, and the optimal pH for this reaction is 7.3 with Co’+ as the divalent cation 8 with Mn2’, and greater than or equal io 8.6 with M8+ (20). Since the optimal pH should be the average of the pK values for water bound to the metal ion (which must be displaced by pyruvate to undergo reaction) and the pK of the lysine residue, we can estimate the pK of the lysine if we know the pK value of water bound to the metal ion on the enzyme. For Co2+, the value of 7.3 from the present study (Fig. 5) suggests that the lysine would also have a pK of 7.3 in the E-Co complex. That is, the pK is reduced by approximately 1.7 pH units by the presence of Co2’, and thus Co2+would bind approximately 50 times more tightly when the lysine is deprotonated; this degree of tightening is consistent with the profile in Fig. apparently fast enough relative to the decarboxylation not to limit V,,. In any case, since CO2release is irreversible, V/K will not be sensitive to this part of the reaction.

OXALACETATE

DECARBOXYLATION

BY PYRUVATE

653

KINASE

TABLE II KINETIC PARAMETERSFOROAA DECARBOXYLATION Nonenzymatic’ (mini)

Metal ion co*+ Mn*+ Mg*+

1.3 0.4 0.3

Enzymeb V (mini)

OAA” K,,, (PM)

Oxalate Ki (PM)

Metal iond Kj bM)

79 51 71

5 20 299

0.02 0.2 20.0”

0.28 0.06 0.35’

’ First-order rate constants determined at pH 5.2 (21). bFrom initial velocity data obtained at pH 7.5 and below where all values of V,, regardless of the metal ion used are pH independent. The reported values were calculated using a subunit molecular weight of 59,250 (3,22), and an enzyme active site concentration calculated using an extinction coefficient at 280 nm for a 1% solution of 5.4 cm-’ (23). ‘From initial velocity data obtained at pH 7.5 and below where all values of Vand V/K, regardless of the metal ion used, are pH independent. d The pH independent value calculated from initial velocity data using Eq. [S]. eDetermined from competitive inhibition vs PEP in the presence of nucleotide (12). fDougherty and Cleland (11).

3. Mn2+ is probably intermediate in its effect on the lysine PK. Dougherty and Cleland (11) have shown that the V and V/K for OAA with Mg+ both decrease above a pK of 9.2, while the Ki (0.02 m&r) for competitive inhibition by oxalate vs PEP is pH independent from pH 5.5-9.0 (12). However, when cobalt is the divalent cation, the V profile decreases above a pK of 8.1, while the V/K for OAA decreases above two pK values with an av-

PKi

8 -0.0, %l 7

1

w PH

FIG. 5. Oxalate inhibition of pyruvate kinase-catalyzed OAA decarboxylation in the presence of Co*‘. The points represent Ki, values calculated from a fit of the data obtained from full inhibition patterns to Eq. [2]. The free [Co*+] was equal to 0.13 mM at pH 7.7 and below, and 0.05 mM at pH 8.2 and above. Uncomplexed oxalate concentrations were equal to 0, Ki, 2Ki, and 4Ki. The l/K, values were fitted to Eq. [5], resulting in the curve shown and a pK value of 7.3 f 0.1.

erage of 7.8 (Fig. 4). From the pKi profile for oxalate in the presence of Co2’ shown in Fig. 5, it can be seen that oxalate binds lOOO-fold tighter to pyruvate kinase than in the presence of Me (12) and the Ki increases above a pK of 7.3. This pK, which presumably represents water coordinated to Co2’, is certainly one of the observed pK values in the V/KOAA profile. Therefore, the other pK value observed in the V/KOAA profile is calculated to be cit. 8.3. The pK value of water coordinated to Me on the enzyme by contrast is 9.3. There is a very large pertubation of this pK (greater than 2 pH units) in going from M8+ to Co2+as expected. Thus, the other pK value observed in both Vand V/K (pK of 8.3 with Co2+ and 9.2 with M8+) is an enzymatic residue which disrupts the charge distribution in the active site and decreases activity when deprotonated. The nature of this group is unknown, but it does not appear to be the catalytic lysine which is involved in enolization, since its pK is a pH unit lower than these values for both Mg2 and Co2+. A summary of the kinetic parameters for oxalacetate decarboxylation by pyruvate kinase is shown in Table II. Also in Table II is a comparison of turnover numbers for the enzymatic and nonenzymatic catalyzed decarboxylation of oxalacetate

654

KIICK

AND

as a function of differing divalent metal ions. ACKNOWLEDGMENTS

The authors express many thanks to Professor G. H. Reed for his many helpful and enthusiastic discussions and to J. Jorgensen for her assistance in acquiring these data.

CLELAND

11. DOUGHERTY,T. Biochemistly 12. DOUGHERTY,T. Biochemistry

M., AND CLELAND, W. W. (1985) 24.5870. M., AND CLELAND, W. W. (1985) 24,5875.

13. MURIHEAD, H., CLAYDEN, D. A., BARFORD, D., LORIMER, C. G., FOTHERGILL-GILMORE, L. A., SCHILTZ, E., AND SCHMITT, W. (1986) EMBO J.

5,475. 14. CLELAND, W. W. (1977) A&V. Enzynol. Relat. Ar-

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