Reaction mechanism for mammalian pyruvate dehydrogenase using natural lipoyl domain substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase com plex (PDC) catalyzes a two-step reaction. Recombinant production of substra te amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) c omponent of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) o r the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 prepara tions, Michaelis constants (K-m) were 52 +/- 14 muM for L1 and 24.8 +/- 3.8 muM for pyruvate and k(cat) was 26.3 s(-1). With less common, higher activ ity preparations of E1, the K-m values were greater than or equal to 160 mu M for L1 and greater than or equal to 35 muM for pyruvate and k(cat) was gr eater than or equal to 70 s(-1). Similar results were found with the L2 dom ain. The best synthetic lipoylated-peptide (L2 residues 163-177) was a much poorer substrate (K-m greater than or equal to 15 mM, k(cat) approximate t o 5 s(-1); k(cat)/K-m decreased >1500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (k(cat)/K-m increased >50 0-fold). Each lipoate source was an effective substrate in the dihydrolipoy l dehydrogenase (E3) reaction, but E3 had a lower K-m for the L2 domain tha n for lipoamide or the lipoylated peptides. In contrast to measurements wit h slow E1 model reactions that use artificial accepters, we con firmed that the natural E1 reaction, using lipoyl domain accepters, was completely inh ibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inh ibited the reverse of the second step catalyzed by E1. The mechanisms by wh ich phosphorylation interferes with E1 activity is interpreted based on acc rued results and the location of phosphorylation sites mapped onto the 3-D structure of related alpha -keto acid dehydrogenases. (C) 2001 Academic Pre ss.