Department of Chemistry, Lomonosov Moscow State University , Moscow, 119991, Russia.
Emanuel Institute of Biochemical Physics, Russian Academy of Sciences , Moscow, 119334, Russia.
J Phys Chem B. 2018 Feb 1;122(4):1378-1386. doi: 10.1021/acs.jpcb.7b10188. Epub 2018 Jan 22.
The transient absorption spectroscopy of hydrolysis of the chromogenic substrate nitrocefin by the L1 metallo-β-lactamase (MβL), a bacterial enzyme responsible for destruction of β-lactam antibiotics molecules, showed formation and decay of a plausible red-shifted reaction intermediate. We propose a mechanism of this important reaction consistent with the transient kinetic data. Quantum mechanics/molecular mechanics (QM/MM) simulations of the reaction pathway revealed occurrence of two reaction intermediates (I1, I2) between the enzyme-substrate (ES) and enzyme-product (EP) complexes. The vertical S-S transition energies calculated at the minimum energy structures (ES, I1, I2, EP) using the time dependent DFT (TD-DFT) method allowed us to assign the experimental absorption bands to all reacting species. We numerically solved the equations of chemical kinetics with the rate constants of all elementary steps evaluated with the transition state theory and simulated the kinetic curves as well as the evolution of the absorption bands of ES, I2, and EP. Direct comparison to the experimental data allowed us to identify the I2 intermediate as the transient red-shifted species detected experimentally. In agreement with the experimental observations, the recomputed energy profiles for the D120N and D120C mutants of L1 reacting with nitrocefin showed absence of a stable intermediate I2. According to the consistent experimental and theoretical results, the breakdown of the intermediate I2 corresponds to the rate-limiting stage of the chemical transformations in the active site of the L1 metallo-β-lactamase. On this basis, we established a QSAR-type correlation between the observed reaction rates (k) of three cephalosporin antibiotics (cefotixin, nitrocefin, cefepime) showing different hydrolysis rates by the L1 metallo-β-lactamase and different structures of the corresponding intermediates of the I2 type. This correlation can be employed for a rational design of novel antibiotics, which are not decomposed by metallo-β-lactamases.
瞬态吸收光谱法研究了一种负责破坏β-内酰胺抗生素分子的细菌酶 L1 金属β-内酰胺酶（MβL）对显色底物硝基头孢菌素的水解反应，结果表明形成并衰减了一种可能的红移反应中间体。我们提出了一个与瞬态动力学数据一致的重要反应机制。反应途径的量子力学/分子力学（QM/MM）模拟揭示了在酶-底物（ES）和酶-产物（EP）复合物之间存在两个反应中间体（I1、I2）。使用含时密度泛函（TD-DFT）方法在最低能量结构（ES、I1、I2、EP）上计算的垂直 S-S 跃迁能使我们能够将实验吸收带分配给所有反应物种。我们用过渡态理论计算了所有基元步骤的速率常数，并对其进行了数值求解，同时还模拟了动力学曲线以及 ES、I2 和 EP 的吸收带的演变。与实验数据的直接比较使我们能够将 I2 中间体识别为实验中检测到的瞬态红移物种。与实验观察结果一致，L1 与硝基头孢菌素反应的 D120N 和 D120C 突变体的重新计算的能量曲线表明，不存在稳定的中间体 I2。根据一致的实验和理论结果，中间体 I2 的分解对应于 L1 金属β-内酰胺酶活性部位中化学转化的限速阶段。在此基础上，我们建立了一个基于观察到的三种头孢菌素抗生素（头孢噻肟、硝基头孢菌素、头孢吡肟）反应速率（k）的定量构效关系（QSAR）型相关性，这些抗生素的水解速率不同，相应的 I2 型中间体的结构也不同。该相关性可用于设计新型抗生素，这些抗生素不会被金属β-内酰胺酶分解。
