Bovine Heart Pyruvate Dehydrogenase Kinase Stimulation by cr-Ketoisovalerate*

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and cr-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic noncompetitive or hyperbolic uncompetitive inhibition at various K’ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 pM over the range of K+ concentrations tested. In the absence of K+, 1 mM a-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K*, cr-ketoisovalerate stimulated PDH kinase activity almost a-fold over the range of O-80 @M thiamin pyrophosphate. Half-maximal stimulation by a-ketoisovalerate occurred at about 200 FM in the presence of 100 pM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 MM thiamin pyrophosphate and 1 mM Nm. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM (Yketoisovalerate were mixed noncompetitive, but cr-ketoisovalerate increased the V,,, and K,,, for adenosine 5’-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove cr-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by cY-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.