Inactivation of human gamma-glutamylcysteine synthetase by cystamine. Demonstration and quantification of enzyme-ligand complexes.
Human erythrocyte gamma-glutamylcysteine synthetase is inactivated by the disulfide cystamine (2,2'-dithiobis-(ethylamine)) at pH 8.2 with a rate constant of 1020 min-1 mM-1. Magnesium ion and various combinations of substrates and products confer differing degrees of protection against cystamine inactivation, thus allowing the detection and quantification of certain enzyme-ligand interactions. By measuring inactivation rates as a function of ligand concentrations in incomplete reaction mixtures, we have obtained evidence for the following complexes: enzyme . Mg2+; enzyme . Mg2+ . MgATP2-; enzyme . Mg2+ . L-glutamate; enzyme . Mg2+ . MgATP2- . L-glutamate; enzyme . Mg2+ . L-gamma-glutamyl-L-alpha-aminobutyrate. The data also imply the existence of enzyme . (Mg2+)2 . MgATP2- . L-glutamate and several enzyme forms resulting from the weak binding to L-alpha-aminobutyrate. The methods used permit the calculation of cystamine inactivation rates for most of these enzyme forms and also give values for the equilibrium constants describing their formation.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000613:Aminobutyrates / Q000378:metabolism; D003538:Cystamine / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors* / Q000097:blood; D005971:Glutamates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*
D000613:Aminobutyrates; D005971:Glutamates; D008024:Ligands; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; D005721:Glutamate-Cysteine Ligase; D008274:Magnesium; D003538:Cystamine