Stole E, Smith T K, Manning J M, Meister A
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
J Biol Chem. 1994 Aug 26;269(34):21435-9.
Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.
阿西维辛（L-(αS,5S)-α-氨基-3-氯-4,5-二氢-5-异恶唑乙酸）对γ-谷氨酰转肽酶的失活作用迅速，被认为是不可逆的，且与每摩尔酶结合近1摩尔抑制剂有关。先前用[3-14C]阿西维辛进行的研究表明，其与大鼠肾酶的一个特定羟基（苏氨酸523）结合（可被底物阻止）。在本研究中，我们发现用羟胺处理被抑制的酶可使这种失活作用逆转。再激活（超过85%完全恢复）与从失活酶中释放出具有苏型-β-羟基-L-γ-谷氨酰异羟肟酸和3-羟基吡咯烷-2-羧酸性质的化合物有关。我们发现该酶对阿西维辛的作用非常缓慢，其速率约为其对谷胱甘肽正常催化速率的10^(-9)，以形成苏型-β-羟基-L-谷氨酸和羟胺。这些发现表明，阿西维辛的抑制作用涉及它在酶上转化为一种抑制性物质，该物质显然通过酯键与酶的一个特定羟基相连。这种中间体释放的极慢速率似乎可以解释所观察到的抑制现象。
