底物结合诱导A类β-内酰胺酶的构象变化，该构象变化会引发催化作用,ACS Catalysis - X-MOL

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底物结合诱导A类β-内酰胺酶的构象变化，该构象变化会引发催化作用
ACS Catalysis ( IF 11.3 ) Pub Date : 2018-02-15 00:00:00 , DOI: 10.1021/acscatal.7b04114
Patricia S. Langan ₁ , Venu Gopal Vandavasi ₁ , Sarah J. Cooper ₂ , Kevin L. Weiss ₁ , Stephan L. Ginell ₃ , Jerry M. Parks _{2,

4} , Leighton Coates ₁

Affiliation

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在临床环境中是一个严重的问题，对于由多药耐药的超级细菌引起的感染，通常留下很少的治疗选择。了解β-内酰胺酶的催化机制对于制定克服耐药性的策略很重要。底物在酶活性位点上的结合可以改变构象和p K _as个催化残基，从而有助于酶催化。在这里，我们报告载脂蛋白形式的A类Toho-1β-内酰胺酶的X射线和中子晶体结构，以及在活动部位带有头孢菌素头孢噻肟的米氏菌类复合物的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的主链会改变其构象，Lys73的Nζ移近保守的催化亲核试剂Ser70的位置。Lys73靠近Ser70的这种移动与酰化之前两个残基之间的质子转移相一致。结合位于Glu166和Ser70位置之间的紧密结合的催化水分子，当Ser70被激活以引起β-内酰胺环的亲核攻击时，该酶被引发催化作用。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Nζ转移到Glu166的Oε2原子比没有质子时更具有热力学优势。综上所述，我们的发现表明底物结合增强了在酰基酶中间体形成之前的初始质子转移步骤的有利性。

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Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis

The emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-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 β-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_as of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-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 Nζ 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 β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 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 acyl-enzyme intermediate.

更新日期：2018-02-15

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