Harrison R K, Stein R L
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Biochemistry. 1990 Feb 20;29(7):1684-9. doi: 10.1021/bi00459a003.
Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond.
亲环蛋白是免疫抑制药物环孢菌素A的胞质结合蛋白，最近已证明它与肽基脯氨酰顺反异构酶相同[菲舍尔，G.，维特曼 - 利博尔德，B.，朗，K.，基费哈伯，T.，& 施密德，F.X.（1989年）《自然》337卷，476页；高桥，N.，早野，T.，& 铃木，M.（1989年）《自然》337卷，473页]。为了为研究亲环蛋白与环孢菌素的相互作用提供一个机制框架，我们研究了肽基脯氨酰顺反异构酶催化的Suc - Ala - Xaa - cis - Pro - Phe - pNA（Xaa = Ala，Gly）从顺式到反式异构化的机制。我们对肽基脯氨酰顺反异构酶的机制研究包括稳态动力学参数的测定、pH和温度依赖性以及溶剂和二级氘同位素效应。这些实验结果支持了一种涉及扭曲催化的机制，即酶利用与底物有利的非共价相互作用释放的自由能来稳定一个以围绕C - N酰胺键的部分旋转为特征的过渡态。
