EVIDENCE FOR 2 LIPOIC ACID RESIDUES PER LIPOATE ACETYLTRANSFERASE CHAIN IN PYRUVATE-DEHYDROGENASE MULTIENZYME COMPLEX OF ESCHERICHIA-COLI

The reaction of 2 maleimides, N-ethylmaleimide and bis-(N-maleimidomethyl) ether, with the pyruvate dehydrogenase multienzyme complex of E. coli in the presence of the substrate, pyruvate, was examined. In both cases the reaction was almost exclusively with the lipoate acetyltransferase [EC 2.3.1.12] component, and the most likely sites of reaction are the lipoic acid residues covalently bound to this component. With both reagents the stoicheiometry of the reaction was measured: 2 mol of reagent reacted with each polypeptide chain of lipoate acetyltransferase, implying that each chain bears 2 functionally active lipoic acid residues. This observation can be reconciled with previous determinations of the lipoic acid content of the complex by allowing for the variability of the subunit polypeptide-chain ratio that can be demonstrated for this multimeric enzyme.