Properties of the nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase from Blastocladiella emersonii

Abstract

The regulatory properties of the NAD-specific isocitrate dehydrogenase ( threo- d s-isocitrate:NAD oxidoreductase (decarboxylating), EC 1.1.1.41) from Blastocladiella emersonii have been studied with steady state kinetic methods. The enzyme was found to catalyze the oxidative decarboxylation of isocitrate as well as the reversed reaction. The results show that the enzyme is extremely sensitive to the buffer concentration in the assay mixture. This inhibition is reversed by the activators, AMP and citrate and is competitive with regard to isocitrate. AMP reduces K m for the substrates but has no effect on V. The enzyme exhibits positive homotropic cooperativity towards isocitrate both in the absence and presence of activators, while the cooperativity towards NAD disappears in the presence of activators as well as high isocitrate concentrations. The activation by AMP occurs according to normal Michaelis Menten kinetics. AMP increases the pH optimum of the reaction. The experimental results are consistent with the possibility that the enzyme contains two binding sites for isocitrate, a catalytic and a regulatory site. Binding of citrate as well as AMP mimics the effect of binding of isocitrate to the regulatory site.