Ivanetich K M, Goold R D
Biomolecular Resource Centre, University of California, San Francisco 94143-0541.
Biochim Biophys Acta. 1989 Sep 14;998(1):7-13. doi: 10.1016/0167-4838(89)90111-8.
Double-reciprocal plots of initial-rate data for the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and GSH by human placental GSH S-transferase pi were linear for both substrates. Computer modelling of the initial-rate data using nonlinear least-squares regression analysis favoured a rapid equilibrium random sequential bi-bi mechanism, over a steady-state random sequential mechanism or a steady-state or rapid equilibrium ordered mechanism. KGSH was calculated as 0.125 +/- 0.006 mM, KCDNB was 0.87 +/- 0.07 mM and alpha was 2.1 +/- 0.3 for the rapid equilibrium random model. The product, S-(2,4-dinitrophenyl)glutathione, was a competitive inhibitor with respect to GSH, and a mixed-type inhibitor toward CDNB (KP = 18 +/- 3 microM). The observed pattern of inhibition is consistent with a rapid equilibrium random mechanism, with a dead-end enzyme.CDNB.product complex, but inconsistent with the inhibition patterns of other bireactant mechanisms. Since rat liver GSH S-transferase 3-3 acts via a steady-state random sequential mechanism [1], while human placental GSH S-transferase and perhaps also rat liver GSH S-transferase 1-1 [2] exhibit rapid equilibrium random mechanisms, we conclude that the kinetic mechanism of the GSH S-transferases is isoenzyme-dependent.
人胎盘谷胱甘肽S-转移酶π催化1-氯-2,4-二硝基苯（CDNB）与谷胱甘肽（GSH）结合反应的初速率数据的双倒数图对两种底物均呈线性。使用非线性最小二乘回归分析对初速率数据进行计算机建模，结果表明，与稳态随机序列机制或稳态或快速平衡有序机制相比，快速平衡随机序列双底物双产物机制更为合适。对于快速平衡随机模型，计算得出KGSH为0.125±0.006 mM，KCDNB为0.87±0.07 mM，α为2.1±0.3。产物S-(2,4-二硝基苯基)谷胱甘肽对GSH而言是竞争性抑制剂，对CDNB而言是混合型抑制剂（KP = 18±3 μM）。观察到的抑制模式与具有终产物酶复合物的快速平衡随机机制一致，但与其他双反应物机制的抑制模式不一致。由于大鼠肝脏谷胱甘肽S-转移酶3-3通过稳态随机序列机制起作用[1]，而人胎盘谷胱甘肽S-转移酶以及可能还有大鼠肝脏谷胱甘肽S-转移酶1-1[2]表现出快速平衡随机机制，我们得出结论，谷胱甘肽S-转移酶的动力学机制是同工酶依赖性的。
