Department of Chemistry, University of California, Davis, California 95616, United States.
Biochemistry. 2013 Aug 27;52(34):5952-65. doi: 10.1021/bi400696j. Epub 2013 Aug 16.
The determination of a complete set of rate constants [free energy profiles (FEPs)] for a complex kinetic mechanism is challenging. Enzymologists have devised a variety of informative steady-state kinetic experiments (e.g., Michaelis-Menten kinetics, viscosity dependence of kinetic parameters, kinetic isotope effects, etc.) that each provide distinct information regarding a particular kinetic system. A simple method for combining steady-state experiments in a single analysis is presented here, which allows microscopic rate constants and intrinsic kinetic isotope effects to be determined. It is first shown that Michaelis-Menten kinetic parameters (kcat and Km values), kinetic isotope efffets, solvent viscosity effects, and intermediate partitioning measurements are sufficient to define the rate constants for a reversible uni-uni mechanism with an intermediate, EZ, between the ES and EP complexes. Global optimization provides the framework for combining the independent experimental measurements, and the search for rate constants is performed using algorithms implemented in the biochemical software COPASI. This method is applied to the determination of FEPs for both alanine racemase and triosephosphate isomerase. The FEPs obtained from global optimization agree with those in the literature, with important exceptions. The method opens the door to routine and large-scale determination of FEPs for enzymes.
确定一套完整的速率常数[自由能谱（FEPs）]对于复杂的动力学机制是具有挑战性的。酶学家设计了各种信息丰富的稳态动力学实验（例如，米氏动力学、动力学参数的粘滞依赖性、动力学同位素效应等），这些实验各自提供了关于特定动力学系统的独特信息。这里提出了一种在单个分析中组合稳态实验的简单方法，该方法允许确定微观速率常数和固有动力学同位素效应。首先表明，米氏动力学参数（kcat 和 Km 值）、动力学同位素效应、溶剂粘滞性效应以及中间分配测量足以定义具有中间 EZ 的可逆单-单机制的速率常数，该中间 EZ 位于 ES 和 EP 复合物之间。全局优化为组合独立的实验测量提供了框架，并且使用在生化软件 COPASI 中实现的算法执行速率常数的搜索。该方法应用于丙氨酸消旋酶和磷酸丙糖异构酶的 FEPs 的确定。通过全局优化获得的 FEPs 与文献中的 FEPs 一致，但存在重要的例外。该方法为酶的 FEPs 的常规和大规模确定开辟了道路。
