Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.

Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffective. Hydrogen peroxide and t-butyl hydroperoxide were inactive. The data indicate pyruvate dehydrogenase kinase (EC 2.7.1.99) contains a thiol group (or groups) that is involved in maintaining a conformation of the enzyme that facilitates phosphorylation and inactivation of its protein substrate, pyruvate dehydrogenase (EC 1.2.4.1). These findings suggest that modulation of pyruvate dehydrogenase kinase activity by thiol-disulfide exchange may be an important physiological mechanism for regulation of kinase activity and, hence, activity of the pyruvate dehydrogenase complex.