Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-β-lactamase from Bacteroides fragilis
Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-β-lactamase from Bacteroides fragilis
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DOI：
10.1021/bi001860v
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发表时间：
2001-02-13
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：
Fast, W;Wang, ZG;Benkovic, SJ
金属- β -内酰胺酶家族的不同成员是在相当大的选择压力下不断发展的临床威胁。蜡样芽孢杆菌酶与脆弱拟杆菌酶在序列、锌化学计量和作用机理上存在差异。为了描绘出更具活性的脆弱芽孢杆菌酶的进化过程，我们对活性位点半胱氨酸残基以及锌含量进行了改变，以模拟蜡样芽孢杆菌酶的变化。具体来说，通过在脆弱芽孢杆菌酶中引入C104R突变，维持了两个锌离子的结合，但使硝基烯烃水解的k（cat）值从226 s（-1）降低到14 s（-1）。从突变体中去除1等量的锌进一步降低k（cat）至4.4 s（-1）。在这两种情况下，观察到的k（cat）非常接近蜡样芽孢杆菌酶的二锌和单锌形式（分别为12和6 s(-1)）。使用硝基芬作为底物的预稳态停流研究表明，这些酶形式具有类似的阴离子中间体的机制，但限速步骤从该物种的质子化转变为导致中间体的C-N键切割。总的来说，已经发现了对C-N键裂解步骤加速贡献3.7 kcal/mol的特征，尽管一些总加速度在稳态中被限速步骤的变化所掩盖。这些实验说明了催化机制进化的一步，从更大的角度来看，也是抗生素耐药机制进化的一步。
The diverse members of the metallo-beta -lactamase family are a growing clinical threat evolving under considerable selective pressure. The enzyme from Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism. To chart the evolution of the more reactive B. fragilis enzyme, we have made changes in an active site cysteine residue as well as in zinc content to mimic that which occurs in the B. cereus enzyme. Specifically, by introducing a C104R mutation into the B. fragilis enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-1). Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-1). In both cases, the observed k(cat) closely approximates that found in the di- and monozinc forms of the B. cereus enzyme (12 and 6 s(-1), respectively). Pre-steady-state stopped-flow studies using nitrocefin as a substrate indicate that these enzyme forms share a similar mechanism featuring an anionic intermediate but that the rate-limiting step changes from protonation of that species to the C-N bond cleavage leading to the intermediate. Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleavage step have been uncovered although some of the total acceleration is masked in the steady-state by a change in rate-limiting step. These experiments illustrate one step in the evolution of a catalytic mechanism and, in a larger perspective, one step in the evolution of antibiotic resistance mechanisms.