Department of Chemistry, University of South Florida , Tampa, Florida 33620, United States.
J Chem Inf Model. 2014 May 27;54(5):1412-24. doi: 10.1021/ci5000517. Epub 2014 May 9.
Benzylpenicillin, a member of the β-lactam antibiotic class, has been widely used to combat bacterial infections since 1947. The general mechanism is well-known: a serine protease enzyme (i.e., DD-peptidase) forms a long lasting intermediate with the lactam ring of the antibiotic known as acylation, effectively preventing biosynthesis of the bacterial cell wall. Despite this overall mechanistic understanding, many details of binding and catalysis are unclear. Specifically, there is ongoing debate about active site protonation states and the role of general acids/bases in the reaction. Herein, a unique combination of MD simulations, QM/MM minimizations, and QM/MM orbital analyses is combined with systematic variation of active site residue protonation states. Critical interactions that maximize the stability of the bound inhibitor are examined and used as metrics. This approach was validated by examining cefoxitin interactions in the CTX-M β-lactamase from E. coli and compared to an ultra high-resolution (0.88 Å) crystal structure. Upon confirming the approach used, an investigation of the preacylated Streptomyces R61 active site with bound benzylpenicillin was performed, varying the protonation states of His298 and Lys65. We concluded that protonated His298 and deprotonated Lys65 are most likely to exist in the R61 active site.
青霉素，β-内酰胺抗生素的一种，自 1947 年以来被广泛用于治疗细菌感染。其作用机制众所周知：一种丝氨酸蛋白酶（即 DD-肽酶）与抗生素的内酰胺环形成持久的中间体，即酰化，有效阻止细菌细胞壁的生物合成。尽管对整体机制有了一定了解，但结合和催化的许多细节仍不清楚。具体来说，关于活性部位质子化状态以及在反应中通用酸/碱的作用仍存在争议。在此，通过将 MD 模拟、QM/MM 最小化和 QM/MM 轨道分析相结合，并系统改变活性部位残基的质子化状态，形成了一种独特的组合。检查了最大化结合抑制剂稳定性的关键相互作用，并将其用作衡量标准。通过检查大肠杆菌 CTX-M β-内酰胺酶中的头孢西丁相互作用并与超高分辨率（0.88 Å）晶体结构进行比较，验证了该方法。在确认使用的方法后，对带有苄青霉素的链霉菌 R61 预酰化活性部位进行了研究，改变了 His298 和 Lys65 的质子化状态。我们得出结论，质子化的 His298 和去质子化的 Lys65 很可能存在于 R61 活性部位。
