Mechanism of Pyruvate Inhibition of Kidney Pyruvate Dehydrogenase, Kinase and Synergistic Inhibition by Pyruvate and ADP*

Pyruvate has been shown to both stimulate and inhibit kidney pyruvate dehydrogenase, (PDH,) kinase activity. The present study investigates the inhibitory effect of pyruvate under conditions in which the stimulatory effect is invariant. Inhibition of PDH, kinase activity by dichloroacetate, a pyruvate analog, is also characterized. Both pyruvate and dichloroacetate are uncompetitive, hyperbolic nonlinear inhibitors with respect to ATP and exhibit synergistic inhibition with ADP, a linear competitive inhibitor with respect to ATP. In the presence of a constant level of ADP, pyruvate or dichloroacetate inhibition of PDH, kinase activity changes to noncompetitive with respect to ATP. In binding studies, pyruvate and dichloroacetate enhance the ability of ADP to competitively inhibit the binding of 13H]ATP to PDH, kinase. Our results indicate that PDH, kinase operates by a sequential (most likely ordered) mechanism in which pyruvate or dichloroacetate does not bind to free PDH, kinase but binds to the E l ADP reaction intermediate. These results show that pyruvate inhibition of PDH, kinase-catalyzed inactivation of the pyruvate dehydrogenase complex is tightly integrated with the level of intramitochondrial ADP and would be greatly enhanced at low ATP:ADP ratios. This linkage may be an important control element in a gluconeogenic tissue such as kidney, in which an elevated pyruvate level should not by itself be an adequate condition for enhancing the activity of the pyruvate dehydrogenase complex.