The relation of the pH and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase.

The relationship of the pH-dependent and concentration-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) was investigated by means of gel filtration chromatography utilizing a standardized Sephacryl S-200 column. The results obtained indicate that the dimeric form of this enzyme dissociates to yield monomers at conditions of low protein concentration or at pH values below neutrality. In addition it is apparent that as the pH is lowered, the minimum concentration of protein required to maintain the enzyme in the dimeric form is increased.     

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.     

Studies on the mechanism of the malate dehydrogenase reaction.

The stereospecificity of the chicken heart mitochondrial malate dehydrogenase as well as the ability of this enzyme to form various abortive complexes has been further investigated. The enzyme was found to be specific for the A-hydrogen of NADH. Complex formation of the enzyme with oxalacetate and oxidized coenzymes is pH-dependent and is promoted at alkaline pH values. The enol form of oxalacetate appears to be the species that participates in the formation of the complexes. The binding of L-malate, D-malate, or hydroxymalonate to the enzyme. NADH complex is also pH-dependent, and involves a group on the enzyme with a pK of 7.5. The binding of L-malate is promoted at alkaline pH values, whereas the binding of D-malate and hydroxymalonate is favored at acidic pH values. These results indicate that L-malate and enol-oxalacetate preferentially or exclusively bind to the nonprotonated form of the enzyme, whereas keto-oxalactate, hydroxymalonate, and D-malate only bind to the protonated form of the enzyme. Based on this conclusion, a detailed chemical mechanism for the malate dehydrogenase reaction has been postulated and a schematic illustration of the transition state of the enzyme is presented.     

Malate dehydrogenases. The lack of evidence for dissociation of the dimeric enzyme in kinetic analyses

The kinetic parameters of beef heart cytoplasmic and pig heart mitochondrial malate dehydrogenases have been examined over a wide range of enzyme concentration. No significant changes are observed in these properties. In conjunction with active enzyme sedimentation and sedimentation equilibrium experiments, it is concluded that there is no evidence for dissociation of the dimeric enzyme at any enyzme level in the kinetic analyses. Thus, if dissociation occurs, it must be too slow to be of significance in determining the kinetic properties of the enzyme. It is shown that unless a subunit and its dimeric form have identical kinetic and substrate binding characteristics, the kinetic parameters should change as a function of enzyme concentration.     

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.     

Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.     

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

Investigation of the relation of the pH-dependent dissociation of malate dehydrogenase to modification of the enzyme by N-ethylmaleimide.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been further characterized using the technique of sedimentation velocity ultracentrifugation. The increased rate and specificity of the inactivation of mitochondrial malate dehydrogenase by the sulfhydryl reagent N-ethylmaleimide has been correlated with the pH-dependent dissociation of the enzyme. Data obtained using NAD+ and its component parts to reassociate the enzyme and also to protect the enzyme from inactivation by N-ethylmaleimide suggest that the sulfhydryl residues being modified by N-ethylmaleimide are inaccessible when the enzyme is in its dimeric form. A dissociation curve for the pH-dependent dissociation suggests that a limited number of residues are being protonated concomitant with dissociation of the enzyme. An apparent pKa of 5.3 has been determined for this phenomenon. Studies using enzyme modified by the sulfhydryl reagent N-ethylmaleimide indicate that selective modification of essential sulfhydryl residues alters the proper binding of NADH.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.     

Isolation and characterization of a manganese-containing superoxide dismutase from yeast.

The cyanide-insensitive superoxide dismutase of yeast has been shown to be localized in the mitochondrial matrix. This enzyme has been isolated in good yield from bakers' yeast. Its molecular weight is 96,000. It is a tetramer, being composed of four subunits of equal size. Exposure to sodium dodecyl sulfate at 100 degrees caused dissociation into dimers, while similar treatment but in the presence of 2-mercaptoethanol caused complete dissociation into monomers. This enzyme contains 1 atom of manganese per subunit and its absorption in the visible suggests Mn(III) in the resting enzyme. Ascorbate caused partial bleaching, presumably by reduction to Mn(II). The amino acid composition was determined. This enzyme has activity comparable to that of other previously reported superoxide dismutases and like the chicken mitochondrial and the bacterial enzymes, its rate of reaction with O2 falls as the pH is raised above 7.8. Crystals of high quality were easily prepared.     

Regulation of mitochondrial malate dehydrogenase: kinetic modulation independent of subunit interaction

Porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37), a dimeric enzyme of Mr = 70,000, is both allosterically activated and inhibited by citrate. Using an affinity elution procedure based upon citrate binding to malate dehydrogenase, the isolation of pure heterodimer (a dimeric species with one active subunit and one iodoacetamide-inactivated subunit) has been achieved. Investigations utilizing this heterodimer in conjunction with resin-bound monomers of malate dehydrogenase have allowed the formulation of a definite conclusion concerning the role of subunit interactions in catalysis and regulation of this enzyme. The citrate kinetic effects, oxaloacetate inhibition, malate activation, and the effects of 2-thenoyl-trifluoroacetone (TTFA) are shown to be independent of interaction between catalytically active subunits. Previous kinetic data thought to support a reciprocating catalytic mechanism for this enzyme may be reinterpreted upon closer analysis in relation to an allosteric, conformationally specific binding model for malate dehydrogenase.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.     

Subunit dissociation of mitochondrial malate dehydrogenase.

Fluorescence polarization studies of porcine mitochondrial malate dehydrogenase labeled with fluorescein isothiocyanate or fluorescamine indicated a concentration-dependent dissociation of the dimeric molecule with a KD OF 2 X 10(7) N at pH 8.0. These results were confirmed by the concentration dependence of the stability of the enzyme at elevated temperatures and the creation of hybrid molecules with fluorescein and Rhodamine B labeled subunits, in which energy transfer was observed. The binding of NADH resulted in a small shift of the subunit dissociation curve toward monomer, demonstrating that monomer has twice the affinity for reduced coenzyme. NAD+ binding prevented dissociation of the dimer, even at concentrations below 10(-8) N. These results indicate that binding of reduced or oxidized coenzymes results in different conformation changes, which are transferred to the subunit interface.     

A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme

The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K(malate) for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K(malate) pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K(malate) indicates the mutant is only active above pH 9. The data suggest that the pK present at about pH 5.6 in all of the pH profiles represents D294, and during catalysis D294 accepts a proton from K199 to allow K199 to act as a general base in the reaction. The pK for the general acid in the reaction is not observed, consistent with rapid tautomerization of enolpyruvate. No other ionizable group in the active site is likely responsible for the partial activity change observed in the pH profiles, and thus the group responsible is probably remote from the active site and the effect on activity is transmitted through the protein by a conformational change.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200-400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.     

NADH binding to porcine mitochondrial malate dehydrogenase.

The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases.

The mechanism that leads to an inhibition of enzyme activity in the presence of high concentrations of substrate was investigated with the two malate dehydrogenase isoenzymes obtained from pig heart. The inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of of oxalacelate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases from chicken hearts. The inhibition constants for oxalacetate are 2.0 mM with the mitochondrial enzyme and 4.5 mM with the cytoplasmic enzyme. Since the in vivo concentration of oxalacetate is reported to be about 10 micrometer, these data suggest that the substrate inhibition that is exhibited by the malate dehydrogenases may not be of any significance in vivo.     

Structure-function relationship of mitochondrial malate dehydrogenase at high dilution and in multicomponent systems

The structure-function relationship of mitochondrial malate dehydrogenase was investigated at low enzyme concentration, as well as in the presence of polyethylene glycol (PEG 6000) and structure making ions. Previous reports claimed the dimeric enzyme to undergo dissociation in dilute solution, and PEG-induced pairing of dimers in the crystalline state. Sedimentation analysis and gel filtration in 0.1 M sodium phosphate pH 7.6 plus 1 mM EDTA and 1 mM dithioerythritol prove the enzyme to be a stable dimer at c greater than or equal to 0.2 microgram/ml (5 nM). In the presence of 8-20% (w/v) PEG 6000, association of the dimer to tetramers and higher aggregates is observed. At 20% (w/v) polyethylene glycol, ultracentrifugal analysis yields up to 50% tetramers; chemical cross-linking by glutaraldehyde confirms the association in a qualitative way. The enzymatic properties of mMDH (specific activity, Km for oxaloacetate and NADH) in the absence and in the presence of PEG 6000 are indistinguishable. At high polyethylene glycol concentrations (greater than or equal to 20%), the thermal stability of the enzyme is found to be increased. The fluorescence emission, as well as the far-UV and near-UV circular dichroism remain unaffected. Accumulated evidence from equilibrium experiments at low enzyme concentration and reconstitution kinetics (after dissociation at acid pH) proves the active species of mMDH to be the dimer.