Gloster Tracey M, Macdonald James M, Tarling Chris A, Stick Robert V, Withers Stephen G, Davies Gideon J
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York, YO10 5YW, United Kingdom.
J Biol Chem. 2004 Nov 19;279(47):49236-42. doi: 10.1074/jbc.M407195200. Epub 2004 Sep 8.
The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.
在寻找新型特效治疗药物以及推导酶的功能和作用机制的过程中，对糖苷水解中过渡态模拟的理解变得越来越重要。为了便于理解，可以通过动力学、热力学和结构分析来表征抑制剂，以构建“抑制谱”。在此，我们剖析了一种四氢恶嗪抑制剂及其衍生物的结合情况，它们的 Ki 值在 500 nM 左右。β-葡萄糖苷酶的 X 射线结构（分辨率为 2 Å）和内切葡聚糖酶的原子分辨率（约 1 Å）揭示了四氢恶嗪抑制剂与这两种酶之间类似的相互作用。动力学分析揭示了两种酶系统中 kcat/Km 和 1/Ki 对 pH 的依赖性，等温滴定量热法揭示了对β-葡萄糖苷酶抑制的焓和熵的贡献。与之前已表征的异法戈明和 1-脱氧野尻霉素的情况不同，两种酶中酶活性对 pH 的依赖性与 1/Ki 对 pH 的依赖性相似。量热分析揭示了一个很大的有利焓，它在结合时被一个不利的熵部分抵消。就 1/Ki 对 pH 的依赖性与 kcat/Km 对 pH 的依赖性的相似谱、与其他糖苷酶抑制剂相比显著的结合焓以及在所测试酶的最佳 pH 下的紧密结合而言，四氢恶嗪及其衍生物是一类比之前设想的要好得多的糖苷酶抑制剂。
