title: On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.
abstract: gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group. Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate. However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol. Thus, the rat kidney and E. coli enzymes share several catalytic features but differ in active site structure. If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.
mesh_terms: D000818:Animals; D001665:Binding Sites; D019328:Buthionine Sulfoximine; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005978:Glutathione / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000494:pharmacology; D010453:Peptide Synthases / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000502:physiology
chemical_list: D013438:Sulfhydryl Compounds; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D010453:Peptide Synthases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione