Jemth P, Mannervik B
Department of Biochemistry, Uppsala University, Biomedical Center, Sweden.
Biochemistry. 1999 Aug 3;38(31):9982-91. doi: 10.1021/bi983065b.
The reaction mechanism of rat glutathione transferase T2-2 has been studied using pre-steady-state and steady-state kinetics. Several parts of the catalytic cycle including binding of substrates, product formation, and product release were investigated. Under saturating conditions, a two-step product release was found to be rate limiting in the enzyme-catalyzed reactions between the nucleophilic substrate glutathione and either of the two electrophilic substrates 1-menaphthyl sulfate and 4-nitrobenzyl chloride. The rate constant for pre-steady-state product formation on rat glutathione transferase T2-2 has an observed pK(a) value of 5.7 apparently due to ionization of the sulfhydryl group of glutathione. This rate constant is approximately 2 orders of magnitude higher than k(cat) at pH values of >6. It can be predicted from the pH dependence that product formation would be the sole rate-limiting step at pH values of <3. A hysteretic mechanism of rGST T2-2 is proposed based on a slow conformational transition detected in pre-steady-state displacement experiments.
已使用预稳态和稳态动力学研究了大鼠谷胱甘肽转移酶T2-2的反应机制。对催化循环的几个部分进行了研究，包括底物结合、产物形成和产物释放。在饱和条件下，发现在亲核底物谷胱甘肽与两种亲电底物1-萘基硫酸酯和4-硝基苄基氯之间的酶催化反应中，两步产物释放是限速步骤。大鼠谷胱甘肽转移酶T2-2上预稳态产物形成的速率常数观察到的pK(a)值为5.7，这显然是由于谷胱甘肽巯基的电离。在pH值>6时，该速率常数比k(cat)高约2个数量级。从pH依赖性可以预测，在pH值<3时，产物形成将是唯一的限速步骤。基于在预稳态置换实验中检测到的缓慢构象转变，提出了rGST T2-2的滞后机制。
