INHIBITION OF MALIC ENZYME FROM GRAPE BERRIES BY SULFHYDRYL-REAGENTS AND OXALIC-ACID

Malic enzyme [malate dehydrogenase (decarboxylating) (NADP+) EC 1.1.1.40] was extracted from Thompson Seedless grape berries and purified 43-fold using DEAE chromatography and affinity chromatography. The enzyme preparation was characterized by SDS PAGE and was shown to contain a major band at 63 kD corresponding to the malic enzyme subunit polypeptide and a minor band of 38 kD. It was demonstrated that the malic enzyme from grape is an EC 1.1.1.40 class enzyme by monitoring pyruvate production from both malate and oxaloacetate using a lactate dehydrogenase assay for pyruvate. The K(m) for malate using manganese as the divalent cation was 0.5 mM which agrees with previous reports, though no allosteric properties were found. Compounds that react with protein sulfhydryl groups were found to affect the activity of grape berry malic enzyme. Monovalent and divalent mercury salts were strong inhibitors; HgCl2 caused complete inhibition at 5-mu-M and p-chloromercuribenzoate gave 72% inhibition at the same concentration. The enzyme was inactivated when treated with 5 mM 5,5'-dithio-bis(2-nitrobenzoic acid), but activity was restored by subsequent treatment with 2 mM dithiothreitol. Oxalate alone at 1 mM also inhibited the enzyme activity but was much more effective in the presence of NADPH.