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首页> 外文期刊>Biochemistry >On the mechanism of the metallo-beta-lactamase from Bacteroides fragilis.
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On the mechanism of the metallo-beta-lactamase from Bacteroides fragilis.

机译：关于脆弱拟杆菌的金属β-内酰胺酶的机理。

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摘要

The catalytic mechanism of metallo-beta-lactamase from Bacteroides fragilis, a dinuclear Zn(II)-containing enzyme responsible for multiple antibiotic resistance, has been investigated by using nitrocefin as a substrate. Rapid-scanning and single-wavelength stopped-flow studies revealed the accumulation during turnover of an enzyme-bound intermediate with intense absorbance at 665 nm (epsilon = 30 000 M(-1) cm(-1)). The proposed minimum kinetic mechanism for the B. fragilis metallo-beta-lactamase-catalyzed nitrocefin hydrolysis [Wang, Z., and Benkovic, S. J. (1998) J. Biol. Chem. 273, 22402-22408] was confirmed, and more accurate kinetic parameters were obtained from computer simulations and fitting. The intermediate was shown to be a novel anionic species bound to the enzyme through a Zn-acyl linkage and contains a negatively charged nitrogen leaving group. This is the first time such an intermediate was observed in the catalytic cycle of a Zn(II)-containing hydrolase and is evidence for a unique beta-lactam hydrolysis mechanism in which the amine can leave as an anion; prior protonation is not required. The electrostatic interaction between the negatively charged intermediate and the positively charged dinuclear Zn(II) center of the enzyme is important for stabilization of the intermediate. The catalytic reaction was accelerated in the presence of exogenous nucleophiles or anions, and neither the product nor the enzyme was modified during turnover, indicating that a Zn-bound hydroxide (rather than Asp-103) is the active site nucleophile. On the basis of all the information on hand, a catalytic mechanism of the B. fragilis metallo-beta-lactamase is proposed.
机译：通过使用硝化甘油作为底物，研究了脆弱拟杆菌（Bacteroides fragilis）的金属-β-内酰胺酶的催化机理，该酶是一种双核含锌（II）酶，对多种抗生素具有抗性。快速扫描和单波长停止流研究表明，在结合酶的中间产物在665 nm（ε= 30 000 M（-1）cm（-1））处有强吸收，在转换过程中会积累。脆弱的芽孢杆菌金属β-内酰胺酶催化的硝基纤维素水解的拟议的最小动力学机理[Wang，Z。，和Benkovic，S.J。（1998）J.Biol.Chem.Soc.Sci。，1998，9，1897]。化学273，22402-22408]得到确认，并且从计算机模拟和拟合获得了更准确的动力学参数。该中间体显示为通过Zn-酰基键与酶结合的新型阴离子物质，并包含带负电荷的氮离去基团。这是首次在含Zn（II）的水解酶的催化循环中发现这种中间体，这是独特的β-内酰胺水解机理的证据，其中胺可作为阴离子离开。不需要事先质子化。带负电的中间体与带正电的双核Zn（II）中心之间的静电相互作用对于稳定中间体非常重要。在外源性亲核试剂或阴离子的存在下，催化反应被加速，产物或酶在周转过程中均未发生修饰，表明与锌结合的氢氧化物（而不是Asp-103）是活性位点亲核试剂。根据现有的所有信息，提出了脆弱脆弱芽孢杆菌金属β-内酰胺酶的催化机理。

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