α-Keto Acid Dehydrogenase Complexes: XX. A KINETIC STUDY OF THE PYRUVATE DEHYDROGENASE COMPLEX FROM BOVINE KIDNEY

Abstract

Abstract The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined. The lipoyl moiety is bound covalently to the transacetylase and, presumably, rotates between the catalytic centers of the three different enzymes that comprise the complex. The kinetic mechanism of the pyruvate dehydrogenase complex from bovine kidney mitochondria has been investigated. Initial velocity patterns were a series of parallel lines, regardless of which substrate was varied at fixed levels of a second substrate. Product inhibition patterns showed that acetyl-CoA is competitive versus CoA and NADH is competitive versus NAD, and that both acetyl-CoA and NADH are uncompetitive versus pyruvate. These results are consistent with the patterns predicted from rate equations derived by Cleland for three-site ping-pong mechanisms. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for acetyl-CoA versus NAD and for NADH versus CoA. Evidence is presented which suggests that these anomalous product inhibition patterns are due to physical association of the flavoprotein with the transacetylase so that combination of acetyl-CoA with the transacetylase hinders combination of NAD with the flavoprotein and combination of NADH with the flavoprotein hinders combination of CoA with the transacetylase.