Institute of Analytical Chemistry, Chemo- and Biosensors , University of Regensburg , Universitätsstr. 31 , 93040 Regensburg , Germany.
J Phys Chem B. 2018 Jun 7;122(22):5809-5819. doi: 10.1021/acs.jpcb.8b02297. Epub 2018 May 23.
Transition-state theory (TST) provides an important framework for analyzing and explaining the reaction rates of enzymes. TST, however, needs to account for protein dynamic effects and heterogeneities in enzyme catalysis. We have analyzed the reaction rates of β-galactosidase and β-glucuronidase at the single molecule level by using large arrays of femtoliter-sized chambers. Heterogeneities in individual reaction rates yield information on the intrinsic distribution of the free energy of activation (Δ G) in an enzyme ensemble. The broader distribution of Δ G in β-galactosidase compared to β-glucuronidase is attributed to β-galactosidase's multiple catalytic functions as a hydrolase and a transglycosylase. Based on the catalytic mechanism of β-galactosidase, we show that transition-state ensembles do not only contribute to enzyme catalysis but can also channel the catalytic pathway to the formation of different products. We conclude that β-galactosidase is an example of natural evolution, where a new catalytic pathway branches off from an established enzyme function. The functional division of work between enzymatic substates explains why the conformational space represented by the enzyme ensemble is larger than the conformational space that can be sampled by any given enzyme molecule during catalysis.
过渡态理论（TST）为分析和解释酶的反应速率提供了一个重要的框架。然而，TST 需要考虑蛋白质动力学效应和酶催化中的异质性。我们通过使用大量纳升级大小的腔室在单分子水平上分析了β-半乳糖苷酶和β-葡糖苷酸酶的反应速率。个体反应速率的异质性提供了关于酶整体中激活自由能（ΔG）的固有分布的信息。与β-葡糖苷酸酶相比，β-半乳糖苷酶的ΔG 分布更广泛，这归因于β-半乳糖苷酶作为水解酶和转糖苷酶的多种催化功能。基于β-半乳糖苷酶的催化机制，我们表明过渡态集合不仅有助于酶催化，还可以引导催化途径形成不同的产物。我们得出结论，β-半乳糖苷酶是自然进化的一个例子，其中新的催化途径从已建立的酶功能分支出来。酶亚基之间的功能分工解释了为什么代表酶整体的构象空间大于任何给定的酶分子在催化过程中可以采样的构象空间。
