Department of Chemistry and Biochemistry , University of Toledo , Toledo , Ohio 43606 , United States.
Department of Chemistry and Chemical Biology , Northeastern University , Boston , Massachusetts 02115 , United States.
Biochemistry. 2018 Apr 24;57(16):2383-2393. doi: 10.1021/acs.biochem.8b00152. Epub 2018 Apr 10.
Tetrahydrolipstatin (THL) is a covalent inhibitor of many serine esterases. In mycobacteria, THL has been found to covalently react with 261 lipid esterases upon treatment of Mycobacterium bovis cell lysate. However, the covalent adduct is considered unstable in some cases because of the hydrolysis of the enzyme-linked THL adduct resulting in catalytic turnover. In this study, a library of THL stereoderivatives was tested against three essential Mycobacterium tuberculosis lipid esterases of interest for drug development to assess how the stereochemistry of THL affects respective enzyme inhibition and allows for cross enzyme inhibition. The mycolyltransferase Antigen 85C (Ag85C) was found to be stereospecific with regard to THL; covalent inhibition occurs within minutes and was previously shown to be irreversible. Conversely, the Rv3802 phospholipase A/thioesterase was more accepting of a variety of THL configurations and uses these compounds as alternative substrates. The reaction of the THL stereoderivatives with the thioesterase domain of polyketide synthase 13 (Pks13-TE) also leads to hydrolytic turnover and is nonstereospecific but occurs on a slower, multihour time scale. Our findings suggest the stereochemistry of the β-lactone ring of THL is important for cross enzyme reactivity, while the two stereocenters of the peptidyl arm can affect enzyme specificity and the catalytic hydrolysis of the β-lactone ring. The observed kinetic data for all three target enzymes are supported by recently published X-ray crystal structures of Ag85C, Rv3802, and Pks13-TE. Insights from this study provide a molecular basis for the kinetic modulation of three essential M. tuberculosis lipid esterases by THL and can be applied to increase potency and enzyme residence times and enhance the specificity of the THL scaffold.
四氢脂霉胺 (THL) 是许多丝氨酸酯酶的共价抑制剂。在分枝杆菌中，已经发现 THL 在处理牛分枝杆菌细胞裂解物时与 261 种脂质酯酶共价反应。然而，由于酶连接的 THL 加合物的水解导致催化周转，在某些情况下，认为共价加合物不稳定。在这项研究中，对一组 THL 立体衍生物进行了测试，这些衍生物针对三种对药物开发有重要意义的结核分枝杆菌脂质酯酶，以评估 THL 的立体化学如何影响各自的酶抑制作用，并允许交叉酶抑制。发现分枝杆菌转移酶抗原 85C (Ag85C) 对 THL 具有立体特异性；共价抑制在数分钟内发生，并且先前已证明是不可逆的。相反，Rv3802 磷脂酶 A/硫酯酶对各种 THL 构型的接受能力更强，并将这些化合物用作替代底物。THL 立体衍生物与聚酮合酶 13（Pks13-TE）的硫酯酶结构域的反应也导致水解周转，并且是非立体特异性的，但发生在较慢的多小时时间范围内。我们的研究结果表明，THL 的β-内酰胺环的立体化学对于交叉酶反应性很重要，而肽臂的两个立体中心可以影响酶特异性和β-内酰胺环的催化水解。所有三种靶酶的观察到的动力学数据都得到了 Ag85C、Rv3802 和 Pks13-TE 的最近发表的 X 射线晶体结构的支持。这项研究的结果为 THL 对三种重要的结核分枝杆菌脂质酯酶的动力学调节提供了分子基础，并可用于提高效力和酶停留时间，增强 THL 支架的特异性。
