IDENTIFICATION OF THE CATALYTIC GLUTAMATE IN THE E1 COMPONENT OF HUMAN PYRUVATE-DEHYDROGENASE

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvat e to acetyl-CoA. The first component (E1) converts pyruvate to bound a cetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors, T here is no 3D structure of E1 available but those of other ThDP-depend ent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha(2)beta(2) struc ture and the conserved Glu(89) Of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expres sed in Escherichia coli and purified. Mutating Glu(89) to glutamine, a spartate and alanine markedly reduces catalytic activity and the affin ity for ThDP, consistent with a role as the catalytic glutamate. (C) 1 998 Federation of European Biochemical Societies.