title: Catalytic mechanism of thioltransferase.
abstract: To evaluate potential catalytic mechanism for thioltransferase thiol-disulfide exchange reactions, seven pig liver mutants were constructed by site-directed mutagenesis. All the expressed enzymes, including wild-type and mutants with the exception of the inactive mutant, ETT-C22S, were variably inhibited by iodoacetamide, and similar results were obtained when these enzymes were preincubated with GSH. However, when preincubated with S-sulfocysteine or hydroxyethyl disulfide, the activity of the enzymes was totally or partially protected against inhibition by iodoacetamide, with the exception of the mutants, ETT-C25S and ETT-C25A. When simultaneously pretreated with GSH and S-sulfocysteine, all enzymes were highly protected. Isoelectric focusing analysis of the above preincubation mixtures showed that different enzyme-substrate intermediates occurred. Using radioactively labeled substrates, [U-14C]cystine and [glycine-2-3H] GSH, enzyme-substrate intermediates were detected. These data indicate that reduced thioltransferase reacts first with disulfide substrates, then with a thiol substrate, e.g. GSH. The formation of either enzyme-substrate mixed disulfide or protein intramolecular disulfide protected the enzyme from inactivation by iodoacetamide. Based on the experimental results, alternative methods of the catalytic mechanism for thioltransferases are proposed.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: Y F
authors.initials: YF
authors.lastname: Yang
authors.forename: W W
authors.initials: WW
authors.lastname: Wells
mesh_terms: D000818:Animals; D001345:Autoradiography; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione); D013552:Swine
chemical_list: D054477:Glutaredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D007460:Iodoacetamide