Tartrate Dehydrogenase Catalyzes the Stepwise Oxidative Decarboxylation of d-Malate with both NAD and Thio-NAD

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Abstract
Tartrate dehydrogenase catalyzes the divalent metal ion- and NAD-dependent oxidative decarboxylation of d-malate to yield CO2, pyruvate, and NADH. The enzyme also catalyzes the metal ion-dependent oxidation of (+)-tartrate to yield oxaloglycolate and NADH. pH−rate profiles and isotope effects were measured to probe the mechanism of this unique enzyme. Data suggest a general base mechanism with likely general acid catalysis in the oxidative decarboxylation of d-malate. Of interest, the mechanism of oxidative decarboxylation of d-malate is stepwise with NAD+ or the more oxidizing thio-NAD+. The mechanism does not become concerted with the latter as observed for the malic enzyme, which catalyzes the oxidative decarboxylation of l-malate [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096−2103]. It appears the change in mechanism observed with malic enzyme is specific to its transition state structure and not a generalized trait of metal ion- and NAD(P)-dependent β-hydroxy acid oxidative decarboxylases. The V/Kmalate pH−rate profile decreases at low and high pH and exhibits pKa values of about 6.3 and 8.3, while that for V/Ktartrate (measured from pH 7.5 to pH 9) exhibits a pKa of 8.6 on the basic side. A single pKa of 6.3 is observed on the acid side of the Vmax pH profile, but the pKa seen on the basic side of the V/K pH profiles is not observed in the Vmax pH profiles. Data suggest the requirement for a general base that accepts a proton from the 2-hydroxyl group of either substrate to facilitate hydride transfer. A second enzymatic group is also required protonated for optimum binding of substrates and may also function as a general acid to donate a proton to the enolpyruvate intermediate to form pyruvate. The 13C isotope effect, measured on the decarboxylation of d-malate using NAD+ as the dinucleotide substrate, decreases from a value of 1.0096 ± 0.0006 with d-malate to 1.00787 ± 0.00006 with d-malate-2-d, suggesting a stepwise mechanism for the oxidative decarboxylation of d-malate. Using thio-NAD+ as the dinucleotide substrate the 13C isotope effects are 1.0034 ± 0.0007 and 1.0027 ± 0.0002 with d-malate and d-malate-2-d, respectively.