Identification of the thiol residues involved in modifications of pig heart lipoamide dehydrogenase by cupric ion and by iodoacetamide

Abstract

The thiol residues involved in two previously described modifications of heart lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) have been identified by comparison of peptide maps of unmodified and modified enzymes. Two thiols and one methionine react when native enzyme is alkylated in concentrated iodoacetamide, with the accompanying loss of enzymatic activity and an NAD-binding site. NAD protects the more slowly reacting thiol from akylation, and the NAD-protected enzyme is active and retains its NAD-binding site. Loss of the binding site, and loss of activity are associated with the alkylation of two neutral thiol peptides which may represent alternative versions of a single thiol region in the enzyme. Treatment of native enzyme with cupric ion results in the rapid oxidation of two thiols to a disulfide bond and loss of NADH-lipoamide reductase activity. We have determined that thiol residues in the cationic peptide and one of the anionic peptides are involved in the disulfide bond formed by cupric ion. Since the cationic peptide contains two histidyl residues, it is proposed that it is the initial site of binding of cupric ion, prior to the oxidation of the thiol residues to a disulfide bond.