Smith T K, Meister A
Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
J Biol Chem. 1995 May 26;270(21):12476-80.
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.
γ-谷氨酰转肽酶是谷胱甘肽代谢中具有核心意义的一种酶，它可被碘乙酰胺灭活，碘乙酰胺会使一个在此被确定为天冬氨酸-422的活性位点羧基发生酯化。用羟胺处理失活的酶会导致去酯化并恢复酶活性。同样能使该酶失活的N-乙酰咪唑会使几个氨基酸残基发生乙酰化。乙酰化会使原本埋藏在天然酶中的半胱氨酸-453暴露出来，从而与碘乙酰胺发生反应。将乙酰化的酶与谷氨酰胺一起温育会产生一种稳定的γ-谷氨酰酶形式，该形式（a）仅位于轻亚基上，（b）对碱比对酸更不稳定，（c）会因用胍离子使酶变性而变得不稳定，并且（d）可与羟胺反应形成γ-谷氨酰异羟肟酸。γ-谷氨酰酶的稳定似乎与赖氨酸残基（包括赖氨酸-99）的乙酰化有关。这些以及其他发现表明，γ-谷氨酰底物的α-氨基通过静电作用与天冬氨酸-422相连，从而促进底物的γ-羰基与酶羟基反应形成γ-谷氨酰酶。
