Reactivity of Thiol Groups in Active and Inactive Forms of a Mitochondrial Nucleoside Diphosphokinase

Incubation of a highly purified, Mg++-dependent nucleoside diphosphokinase (EC 2.7.4.6) of bovine liver mitochondria with p-chloromercuriphenylsulfonate (CMS) caused activation at low concentrations and inactivation at higher concentrations of CMS. Inactivation was greatly accelerated in the presence of Mg++, suggesting that Mg++ stabilizes a form of the enzyme in which essential thiol groups are more reactive to CMS. MgADP, on the other hand, completely prevented inactivation of the enzyme by CMS. Free ADP, ATP, and MgATP alone gave slight protection, and AMP, flavin mononucleotide, flavin adenine dinucleotide, NAD+, NADP+, and pyrophosphate were completely ineffective. These findings indicate that MgADP favors an equilibrium form of the enzyme with diminished reactivity of essential thiol groups. They are also consistent with earlier studies (Goffeau, A., Pedersen, P. L., and Lehninger, A. L., J. Biol. Chem., 242, 1845 (1967)) showing that MgADP is a potent inhibitor of enzyme activity. Magnesium uridine diphosphate and magnesium cytidine diphosphate also protected against inactivation by CMS in proportion to their effectiveness as inhibitors of the enzymatic reaction. The apparent binding constants of MgADP, either as substrate or inhibitor, remained unchanged in the presence of CMS, suggesting that the mercurial is able to react selectively with one form of the enzyme while leaving the other form completely active. Addition of 2-mercaptoethanol caused activation at low concentrations, but at high concentrations produced an inhibited state of the enzyme. MgADP did not protect against the inhibitory effect of mercaptoethanol. From these experiments, at least three equilibrium forms of the enzyme can be recognized: an active form stabilized by Mg++, an inhibited form stabilized by MgADP, and an inactive form in which the essential thiol group (or groups) is oxidized.