Gadda Giovanni, Sobrado Pablo
Department of Biochemistry , Virginia Tech , Blacksburg , Virginia 24061 , United States.
Biochemistry. 2018 Jun 26;57(25):3445-3453. doi: 10.1021/acs.biochem.8b00232. Epub 2018 Jun 13.
The study of enzyme reaction mechanisms is fundamentally important to our understanding of biochemistry, cellular metabolism, and drug development. This Perspective focuses on the use of kinetic solvent viscosity effects (KSVEs) to study enzyme reactions. This technique is easily implemented and uses steady-state kinetic analyses to probe whether substrate binding is diffusion-controlled and whether product release is the rate-limiting step in the catalytic cycle. In addition, KSVEs can identify isomerization steps that are important for catalysis. The use of KSVEs in combination with other techniques, such as kinetic isotope effects, pH effects, and site-directed mutagenesis, can provide a detailed view of the mechanism of enzyme action. We present the basic theory, important experimental considerations, and potential outcomes and briefly discuss some examples from the literature. The derivation of the equations that are important for data analysis is also presented.
酶反应机制的研究对于我们理解生物化学、细胞代谢和药物开发至关重要。本综述聚焦于利用动力学溶剂粘度效应（KSVEs）来研究酶反应。该技术易于实施，采用稳态动力学分析来探究底物结合是否受扩散控制以及产物释放是否为催化循环中的限速步骤。此外，KSVEs 能够识别对催化作用至关重要的异构化步骤。将 KSVEs 与其他技术（如动力学同位素效应、pH 效应和定点诱变）结合使用，可以提供酶作用机制的详细视图。我们阐述了基本理论、重要的实验考量因素以及潜在结果，并简要讨论了文献中的一些实例。还给出了对数据分析至关重要的方程的推导。
