SUMMARY: A soluble, NAD-dependent D(-)-mandelate dehydrogenase has been purified to homogeneity from the yeast strain KGX 39, using DEAE-Sephacel, Phenyl Sepharose and Matrex Gel Orange A affinity chromatography. The of the native enzyme was 77000 (as determined by gel filtration) and the subunit was 38000 (as determined by SDS-polyacrylamide-gel electrophoresis), indicating that the enzyme exists as a dimer. Amino acid analysis showed only one cysteine residue per subunit. There was no spectroscopic evidence for the presence of cofactors such as flavin or cytochrome. The enzyme was neither inhibited nor stimulated by a wide range of salts and metal ions, nor was it inhibited by various metal chelating agents, indicating that the enzyme has no absolute requirement for salt or metal ions. The enzyme was not inhibited by various thiol reagents. The isoelectric point was 5·9. (-)-Mandelate dehydrogenase catalyses the oxidation of (-)-mandelate (forward reaction) and the reduction of phenylglyoxylate (reverse reaction). Activities of the forward reaction and reverse reactions were maximal at pH 9·5 and pH 5·85 respectively. Oxidation of (-)-mandelate produced equimolar amounts of phenylglyoxylate and NADH, and the equilibrium constant was 1·59 x 10 , pH 9·2. At pH 9·5, the values for (-)-mandelate and NAD were 319 μM and 71 μM respectively, and the maximum velocity was 123 μmol min (mg of protein). The apparent and values with respect to (-)-mandelate, NAD, phenylglyoxylate and NADH at pH5·85, pH 70 and pH 9·5 are recorded. (-)-Mandelate dehydrogenase uses various substituted mandelates as substrates. Several aliphatic 2-hydroxy and 2-oxocarboxylic acids did not act as substrates but were capable of inhibiting the enzyme. Comparisons are made with other mandelate dehydrogenases and NAD-dependent dehydrogenases.


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