Chemical modification of rat liver cytosolic NADP(+)-linked isocitrate dehydrogenase by N-ethylmaleimide. Evidence for essential sulphydryl groups.

Incubation of rat liver cytosolic isocitrate dehydrogenase with N-ethylmaleimide (NEM) resulted in the inactivation of the enzyme following pseudo-first order kinetics. Isocitrate affords considerable protection against inactivation whereas NADP+ enhances modification of the enzyme, suggesting localization of the modified group at the active site. Correlation of loss of activity with incorporation of [14C]NEM indicated that two sulphydryl residues/sub-unit are modified of which only one is shown to be involved in catalysis. pH dependence of the inactivation process implicates a reactive group of pKa 8.1 in catalysis. We conclude that a unique cysteine residue is essential for maximal catalytic activity of isocitrate dehydrogenase.