Intermediate partitioning in the tartrate dehydrogenase-catalyzed oxidative decarboxylation of D-malate.

The oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase has been examined in detail. Enzyme-catalyzed partitioning of oxalacetate has been determined to proceed with formation of pyruvate and D-malate in a ratio of 3.7 to 1. These data, along with the deuterium and tritium kinetic isotope effects on hydride transfer, allow exact calculation of the intrinsic isotope effect and the forward and reverse commitments to catalysis, which have values of 5.1 +/- 0.8, 6.3 +/- 1.0, and 2.0 +/- 0.3, respectively. The viscosity dependence of the tritium isotope effect was measured, which allowed determination of the internal and external components of the commitment factors. The reverse commitment has no external portion, but the forward commitment can be divided into external and internal portions of 3.7 +/- 1.2 and 2.6 +/- 1.6, respectively. These data indicate that the reaction becomes committed to catalysis in the forward direction by formation of the Michaelis complex; reverse hydride transfer from NADH to OAA is twice as fast as decarboxylation of OAA, and recarboxylation of pyruvate occurs at a negligible rate. The rate constant for dissociation of OAA from the enzyme active site was estimated to be approximately 4 orders of magnitude slower than that for dissociation of oxaloglycolate, which is the product of the enzyme-catalyzed oxidation of (+)-tartrate.