Ren Weiwu, Farren-Dai Marco, Sannikova Natalia, Świderek Katarzyna, Wang Yang, Akintola Oluwafemi, Britton Robert, Moliner Vicent, Bennet Andrew J
Department of Chemistry, Simon Fraser University Burnaby British Columbia V5A 1S6 Canada
Departament de Química Física i Analítica, Universitat Jaume I 12560 Castellón Spain
Chem Sci. 2020 Sep 16;11(38):10488-10495. doi: 10.1039/d0sc04401f.
Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds an exploded, or loose, S2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds.
碳环糖是天然存在的碳水化合物的结构类似物，能够与参与碳水化合物加工的酶相互作用并抑制这些酶。特别是，碳环糖作为糖苷水解酶（GHs）的抑制剂以及在多个疾病领域作为治疗先导物而受到关注。然而，目前尚不清楚碳环糖是如何被GHs识别和加工的。在此，我们报道了三种碳环糖同位素异构体的合成，并结合实验测量的动力学同位素效应和QM/MM混合计算，对初始GH - 碳环糖共价中间体的形成以及该中间体的水解进行了详细的过渡态（TS）分析。我们发现，来自[具体来源未提及]的α - 半乳糖苷酶能有效地稳定在一个与反应的碳环糖协同作用的远程C5 - 烯丙基中心上的TS电荷发展，并且催化过程通过一个扩展的或松散的S2过渡态进行，没有离散的酶结合阳离子中间体。我们得出结论，除了我们对酶 - 天然底物复合物的TS结构的了解之外，关于酶与非天然碳环糖底物反应的TS结构的知识表明，GHs能够稳定比以前认为的更广泛的带正电TS结构。此外，这种更深入的理解将有助于设计新的碳环糖GH过渡态类似物，例如用作化学生物学工具和药物先导化合物。
