Transient-state kinetic analysis of the oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase.
The oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase has been analyzed by transient-state kinetic methods and kinetic isotope effect measurements. The reaction time courses show a burst of NADH formation prior to the attainment of the steady-state velocity. The binding of the inhibitor tartronate to the enzyme was examined by monitoring the quenching of the protein's intrinsic fluorescence; the tartronate concentration dependence of the observed rate constant for association was hyperbolic, supporting a two-step model for inhibitor binding. Analysis of the time courses for D-malate oxidation yielded values for many of the microscopic rate constants governing the reaction. The range of possible solutions for the microscopic rate constants was constrained by comparison of the time course for oxidation of unlabeled malate with that of deuterated malate; this analysis relied on the determination of the intrinsic isotope effect on hydride transfer via measurement of D(V/K), T(V/K), and the oxaloacetate partition ratio. The results of the transient-state kinetic analyses suggest that the rate of D-malate oxidation is largely limited by the rate of decarboxylation of the intermediate oxaloacetate which occurs at 11 s-1. Hydride transfer from D-malate to NAD+ occurs with a rate constant of 300 s-1, and (D)k for this step is 5.5. The agreement between experimentally measured steady-state kinetic parameters and kinetic isotope effects and their values calculated from the microscopic rate constants derived from the transient-state kinetic analyses was quite good.
Tipton
P A
PA
Department of Biochemistry, University of Missouri--Columbia, 65211, USA.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003653:Decarboxylation; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008293:Malates / Q000378:metabolism*; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013646:Tartronates / Q000378:metabolism
D008293:Malates; D011994:Recombinant Proteins; D013646:Tartronates; C032973:tartronic acid; C030298:malic acid; D000429:Alcohol Oxidoreductases; C038481:tartrate dehydrogenase