催化反应的机制mandelate消旋酶:K166R突变体的结构和机械性能。
文章的细节
-
引用
复制到剪贴板 -
Kallarakal, Mitra B, Kozarich JW, Gerlt是的,克利夫顿詹,Petsko GA,肯扬GL
催化反应的机制mandelate消旋酶:K166R突变体的结构和机械性能。
生物化学,1995年3月7日,34 (9):2788 - 97。
- PubMed ID
-
7893690 (在PubMed]
- 文摘
-
的基础上可用的高分辨率结构mandelate消旋酶(先生)从假单胞菌putida [Landro, j。Gerlt, j。Kozarich, j·W。古,c W。沙,诉J。肯扬,g . L。、Neidhart d J。藤田,J。& Petsko g . a(1994)生物化学33岁,635 - 643],赖氨酸166年和297年定位适当参与催化酸/碱催化剂,抽象的对映体的质子mandelate形成烯醇的中间体或使质子化烯醇的中间体形成mandelate的对映体,与赖氨酸166参与(S)特殊酸/碱催化剂,他297年参加(R)特殊酸/碱催化剂。在本文中,我们报告的结构和机械性能的突变体166赖氨酸与精氨酸(K166R)所取代。K166R的结构已经确定在1.85决议与衬底(S) -mandelate绑定在活动网站。这个复杂的结构揭示了没有几何变化活跃的网站,除了Arg的长侧链166年必然是流离失所的向上的位置被赖氨酸166年由立体交互与衬底。与H297N [Landro, j .先生的突变。Kallarakal, a . T。赎金,s . C。Gerlt, j。, Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9275-9281], the K166R exhibits low levels of racemase activity [kcat is reduced 5 x 10(3)-fold in the (R)- to (S)-direction and 1 x 10(3)-fold in the (S)- to (R)-direction]. The substrate and solvent deuterium isotope effects support a reaction coordinate for the K166R-catalyzed reaction in which the transition state for interconversion of bound (S)-mandelate and the stabilized enolic intermediate is higher in energy that the transition state for interconversion of bound (R)-mandelate and the stabilized enolic intermediate. The solvent deuterium isotope effect when (S)-mandelate is substrate (2.2 +/- 0.3) supports the proposal that the formation of the enolic intermediate involves partial transfer of a solvent-derived proton from Glu 317 to the substrate as the alpha-proton is abstracted [Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2777-2787].(ABSTRACT TRUNCATED AT 400 WORDS)