Chemical modification of active site residues in gamma-glutamyl transpeptidase. Aspartate 422 and cysteine 453.
gamma-Glutamyl transpeptidase, an enzyme of central significance in glutathione metabolism, is inactivated by iodoacetamide, which esterifies an active site carboxyl group identified here as that of Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads to deesterification and to restoration of enzymatic activity. N-Acetylimidazole, which also inactivates the enzyme, acetylates several amino acid residues. Acetylation exposes Cys-453, which is buried in the native enzyme, to reaction with iodoacetamide. Incubation of the acetylated enzyme with glutamine produces a stabilized gamma-glutamyl-enzyme form which is (a) located exclusively on the light subunit, (b) more labile to base than to acid, (c) destabilized by denaturation of the enzyme with guanidinium ions, and (d) reactive with hydroxylamine to form gamma-glutamylhydroxamate. Stabilization of the gamma-glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the alpha-amino group of the gamma-glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the gamma-carbonyl of the substrate with an enzyme hydroxyl group to form a gamma-glutamyl-enzyme.
Smith
T K
TK
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
Meister
A
A
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
D006898:Hydroxylamines; D007093:Imidazoles; D019811:Hydroxylamine; D001224:Aspartic Acid; D005723:gamma-Glutamyltransferase; C012160:N-acetylimidazole; D007460:Iodoacetamide