Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for essential histidine residue at the active site of lipoamide dehydrogenase

Abstract

Incubation of pyruvate dehydrogenase multienzyme ocmplex (PD complex) from E. coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial anchoring of the reagent via its AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2, EC 2.3.1.12) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subuntis were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of .apprx. 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.