Bilevel disulfide group reduction in the activation of c(4) leaf nicotinamide adenine dinucleotide phosphate-malate dehydrogenase.

The time course of thioredoxin-mediated reductive activation of isolated Zea mays nicotinamide adenine dinucleotide phosphatemalate dehydrogenase is highly sigmoidal in nature. We examined the factors affecting these kinetics, including the thiol-disulfide status of unactivated and activated forms of the enzyme. The maximum steady rate of activation was increased, and the length of the lag in activation decreased, as the concentrations of thioredoxin-m, dithiothreitol, and KCl were increased. The lag in activation (sigmoidicity) was eliminated by preincubating the unactivated enzyme with 100 mm 2-mercaptoethanol; this pretreatment did not activate the enzyme. Unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase was found to contain approximately two SH groups per subunit, increasing to about four SH per subunit after pretreatment with 2-mercaptoethanol and six SH per subunit after activation by incubating the enzyme with dithiothreitol. We suggest that reduction of one particular higher redox potential disulfide group in unactivated nicotinamide adenine dinucleotide phosphate-malate dehydrogenase facilitates the subsequent reduction of the critical S-S group (regulatory S-S) necessary to generate the active form of the enzyme.