Studies on the Active Site of γ-Glutamyltransferase

Summary

γ-Glutamyltransferase (E.G.2.3.2.2) from rat kidney catalyzes the hydrolysis of glutathione via a covalent γ-Glutamyl-enzyme intermediate. The identity of some active site amino acid residues and of the residue which is covalently attached to the γ-Glutamyl group was investigated by means of chemical modification. The enzyme was not inhibited by p-hydroxymercuribenzoate or by phenylmethylsulfonyl fluoride, implying the absence of active site cysteine or serine residues, but a cysteine residue was nonetheless later shown to be close to the active site. Inhibition by carbodiimide suggested the presence of an active site COOH group, and by trinitrobenzene sulfonic acid suggested that a lysine residue was important. Reaction of the active site with [¹⁴C] iodoacet-amide, in conjunction with N-acetylimidazole treatment, led to labeling both of a COOH group and of a cysteine residue. Treatment of the enzyme with N-acetylimidazole modified a lysine residue, and inhibited the enzyme but did not block formation of the γ-Glutamyl-enzyme intermediate. This lysine residue when not acetylated appears to protect the cysteine residue from reaction with iodoacetamide. The γ-Glutamyl group in the covalent enzyme intermediate is stable to performid acid, and to 1 M hydroxylamine in 6 M urea at pH 9, but is removed slowly at pH 12, suggesting that the γ-Glutamyl residue must be attached to a second lysine residue by an amide linkage. This isopeptide linkage is known from studies on fibrin cross-linking, and attempts are under way to isolate γ-[¹⁴C] glutamyl-ε-lysine from the γ-Glutamyl-enzyme intermediate.