Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
J Am Chem Soc. 2010 May 12;132(18):6474-80. doi: 10.1021/ja1007849.
A light-activated reaction analog has been developed to mimic the catalytic reaction cycle of Delta(5)-3-ketosteroid isomerase to probe the functionally relevant protein solvation response to the catalytic charge transfer. Delta(5)-3-ketosteroid isomerase from Pseudomonas putida catalyzes a C-H bond cleavage and formation through an enolate intermediate. Conversion of the ketone substrate to the enolate intermediate is simulated by a photoacid bound to the active site oxyanion hole. In the ground state, the photoacid electrostatically resembles the enolate intermediate while the low pK(a) excited state resembles the ketone starting material. Time-resolved fluorescence experiments with photoacids coumarin 183 and equilenin show the active site of Delta(5)-3-ketosteroid isomerase to be largely unperturbed by the light-activated reaction. The small solvation response for the photoacid at the active site as compared with a simple solvent suggests the active site does not significantly change its electrostatic environment during the catalytic cycle. Instead, the reaction takes place in an electrostatically preorganized environment.
已开发出一种光激活反应类似物，以模拟 Delta(5)-3-酮固醇异构酶的催化反应循环，从而探究与功能相关的蛋白质溶剂化对催化电荷转移的响应。假单胞菌属中的 Delta(5)-3-酮固醇异构酶通过烯醇化物中间体催化 C-H 键的断裂和形成。酮底物向烯醇化物中间体的转化通过与活性位点氧阴离子穴结合的光酸模拟。在基态下，光酸在静电上类似于烯醇化物中间体，而低 pK(a)激发态类似于酮起始物质。用光酸香豆素 183 和 Equilenin 进行的时间分辨荧光实验表明，Delta(5)-3-酮固醇异构酶的活性位点受光激活反应的影响很小。与简单溶剂相比，光酸在活性位点的小溶剂化响应表明，在催化循环过程中，活性位点的静电环境不会发生显著变化。相反，反应发生在静电预组织的环境中。
