Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis
Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis
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DOI：
10.1021/acscatal.7b04114
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发表时间：
2018-03-01
期刊：
影响因子：
12.9
通讯作者：
Coates, Leighton
中科院分区：
文献类型：
--
作者：
Langan, Patricia S.;Vandavasi, Venu Gopal;Coates, Leighton
通过β-内酰胺酶产生和传播细菌对β-内酰胺抗生素的耐药性是临床环境中的一个严重问题，通常对多重耐药超级细菌引起的感染几乎没有治疗选择。了解 β-内酰胺酶的催化机制对于制定克服耐药性的策略非常重要。酶活性位点中底物的结合可以改变催化残基的构象和 pK(a)，从而有助于酶催化。在这里，我们报告了 apo 形式的 A 类 Toho-1 β-内酰胺酶的 X 射线和中子晶体结构，以及活性位点与头孢菌素抗生素头孢噻肟的米氏复合物的 X 射线结构。这些结构的比较表明底物结合引起一系列变化。在催化中重要的保守残基 Lys73 和 Tyr105 的侧链以及 Ser130 的主链改变了它们的构象，Lys73 的 N zeta 移近了保守催化亲核试剂 Ser70 的位置。 Lys73 更接近 Ser70 的这种移动与酰化之前两个残基之间的质子转移一致。与位于 Glu166 和 Ser70 位置之间紧密结合的催化水分子相结合，当 Ser70 被激活以对 β-内酰胺环进行亲核攻击时，该酶就准备好进行催化。野生型酶模型的量子力学/分子力学 (QM/MM) 自由能模拟表明，质子从 Lys73 的 N zeta 转移到 Glu166 的 O epsilon 2 原子在热力学上比不存在时更有利。综上所述，我们的研究结果表明，底物结合增强了芳基酶中间体形成之前的初始质子转移步骤的有利性。
The emergence and dissemination of bacterial resistance to beta-lactam antibiotics via beta-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of beta-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and pK(a)s of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 beta-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with N zeta of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 is activated for nucleophilic attack of the beta-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the N zeta of Lys73 to the O epsilon 2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the aryl-enzyme intermediate.