On the mechanism of the metallo-β-lactamase from Bacteroides fragilis
On the mechanism of the metallo-β-lactamase from Bacteroides fragilis
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DOI：
10.1021/bi990356r
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发表时间：
1999-08-03
期刊：
影响因子：
2.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Wang, ZG;Fast, W;Benkovic, SJ
脆弱拟杆菌中的金属-β-内酰胺酶是一种双核 Zn(II) 酶，可导致多种抗生素耐药性，已使用头孢硝酯作为底物研究了其催化机制。快速扫描和单波长停流研究揭示了酶结合中间体在周转过程中的积累，该中间体在 665 nm（ε = 30 000 M-1 cm(-1)）处具有强烈吸光度。所提出的脆弱芽孢杆菌金属-β-内酰胺酶催化 cm 头孢硝基水解的最小动力学机制 [Wang, Z., and Benkovic, S. J. (1998) J. Biol.化学。 273, 22402-22408]得到证实，并通过计算机模拟和拟合获得了更准确的动力学参数。该中间体被证明是一种新型阴离子物质，通过锌-酰基键与酶结合，并含有带负电的氮离去基团。这是首次在含 Zn(II) 的水解酶的催化循环中观察到此类中间体，并且是独特的 β-内酰胺水解机制的证据，其中胺可以作为阴离子离开；不需要预先质子化。带负电的中间体和酶的带正电的双核 Zn(II) 中心之间的静电相互作用对于中间体的稳定很重要。在外源亲核试剂或阴离子存在的情况下，催化反应加速，并且产物和酶在周转过程中都没有被修饰，表明锌结合的氢氧化物（而不是Asp-103）是活性位点亲核试剂。根据现有的所有信息，提出了脆弱拟杆菌金属-β-内酰胺酶的催化机制。
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 cm 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.