Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β-Lactamase from Mycobacterium tuberculosis
Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β-Lactamase from Mycobacterium tuberculosis
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DOI：
10.1021/bi400177y
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发表时间：
2013-06-11
期刊：
影响因子：
2.9
通讯作者：
Blanchard, John S.
中科院分区：
文献类型：
--
作者：
Chow, Carmen;Xu, Hua;Blanchard, John S.
组成型表达的染色体编码的β-内酰胺酶（BlaC）是负责结核分枝杆菌中对β-内酰胺抗生素的内在耐药性的酶。来自该实验室的先前研究已经表明，该酶表现出广谱表型，具有非常高水平的青霉素酶和头孢菌素酶活性，以及弱的碳青霉烯酶活性[Tremblay，L. W.的，等人（2008）Biochemistry 47，5312-5316]。在这份报告中，我们已经确定了pH值的动力学参数的依赖性，揭示了最大速度取决于电离状态的两个组：一个一般的碱表现出pK值为4.5和一个一般的酸表现出pK值为7.8。在定义了动力学参数与pH无关的区域（pH 6.5）后，我们确定了k（cat）值相差5.5个数量级的三种底物的溶剂动力学同位素效应（SKIE）。硝头孢菌素是一种高度活化的显色头孢菌素衍生物，对V和V/K均表现出1.4的稳态溶剂动力学同位素效应。头孢西丁是一种较慢的头孢菌素衍生物，在稳态实验中，其对V的SKIE为3.9，但对V/K的SKIE为1.8。头孢西丁的前稳态停流实验显示，β-内酰胺开环爆发，酰化步骤的SKIE值为1.6，脱酰步骤的SKIE值为3.4。美罗培南是BlaC的一种非常慢的底物，在稳态实验中表现出爆发动力学。美罗培南的SKIE测定显示，酰化和脱酰步骤的SKIE分别为3.8和4.0。在所有情况下的质子库存是线性的，表明一个单一的溶剂衍生的质子在化学步骤负责SKIE的参与。这些底物的β-内酰胺水解的限速步骤进行了分析，并负责观察到的SKIE的化学步骤进行了讨论。
The constitutively expressed, chromosomally encoded beta-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to beta-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose k(cat) values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of beta-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for beta-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.