Wang Z, Fast W, Benkovic S J
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1999 Aug 3;38(31):10013-23. doi: 10.1021/bi990356r.
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.
以硝基头孢菌素为底物，对脆弱拟杆菌金属β-内酰胺酶（一种含双核锌（II）且导致多重抗生素耐药性的酶）的催化机制进行了研究。快速扫描和单波长停流研究表明，在反应过程中积累了一种与酶结合的中间体，该中间体在665 nm处有强烈吸收（ε = 30 000 M⁻¹ cm⁻¹）。脆弱拟杆菌金属β-内酰胺酶催化硝基头孢菌素水解的最小动力学机制（Wang, Z., and Benkovic, S. J. (1998) J. Biol. Chem. 273, 22402 - 22408）得到了证实，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通过锌-酰基键与酶结合的新型阴离子物种，含有一个带负电荷的氮离去基团。这是首次在含锌（II）水解酶的催化循环中观察到这样的中间体，这为一种独特的β-内酰胺水解机制提供了证据，即胺可以作为阴离子离去；不需要预先质子化。带负电荷的中间体与酶带正电荷的双核锌（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下，催化反应加速，并且在反应过程中产物和酶均未被修饰，这表明与锌结合的氢氧化物（而非天冬氨酸103）是活性位点亲核试剂。基于现有的所有信息，提出了脆弱拟杆菌金属β-内酰胺酶的催化机制。
