Mammalian Pyruvate Dehydrogenase Complex Research Articles

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

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.

Function of calcium ions in pyruvate dehydrogenase phosphatase activity

Phosphorylation and dephosphorylation of the pyruvate dehydrogenase (PDH) component of the mammalian pyruvate dehydrogenase complex are catalyzed, respectively, by a MgATP 2-requiring kinase and a Mg 2+-requiring phosphatase. The kinase and the PDH, but not the phosphatase, are tightly bound to the dihydrolipoyl transacetylase core of the complex. Evidence is presented that the activity of the PDH phosphatase from bovine kidney and heart is increased about tenfold when it is attached to the transacetylase. Ca 2+ is required to bind the phosphatase, but not the kinase or the PDH, to the transacetylase, thereby facilitating the Mg 2+-dependent dephosphorylation of the phosphorylated PDH. Ca 2+ lowers the apparent K m of the phosphatase for phosphorylated PDH about twentyfold. The Ca 2+-controlled association of the phosphatase and the transacetylase could provide an important mechanism for regulation of the phosphorylation-dephosphorylation cycle.

α-Keto acid dehydrogenase complexes: XVI. Studies on the subunit structure of the pyruvate dehydrogenase complexes from bovine kidney and heart

The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (a flavoprotein) are joined. Electron microscopic studies indicated that the design of the transacetylase is based on icosahedral (532) symmetry and, therefore, it would be expected to contain 60 very similar, if not identical, polypeptide chains. Evidence is presented that the bovine kidney and heart dihydrolipoyl transacetylases are very similar and that each enzyme does indeed consist of 60 apparently identical polypeptide chains. Each chain apparently contains 1 molecule of covalently bound lipoic acid. The molecular weight of the transacetylase is about 3.12 ± 0.20 million. The pyruvate dehydrogenase components of the bovine kidney and heart pyruvate dehydrogenase complexes are also very similar. The uncomplexed enzymes have a molecular weight of about 154,000 and possess the subunit structure α 2 β 2. The molecular weights of two subunits are about 41,000 and 36,000, respectively. Only the α-subunit undergoes phosphorylation in the presence of pyruvate dehydrogenase kinase and ATP. Possible functions of the two subunits are discussed. It appears that the bovine kidney and heart pyruvate dehydrogenase complexes contain about 60 pyruvate dehydrogenase units (αβ) but only 10–12 flavoprotein chains.