Schneider Samuel H, Kozuch Jacek, Boxer Steven G
Chemistry Department, Stanford University, Stanford, California 94305, United States.
ACS Cent Sci. 2021 Dec 22;7(12):1996-2008. doi: 10.1021/acscentsci.1c00880. Epub 2021 Nov 22.
The interplay of enzyme active site electrostatics and chemical positioning is important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 β-lactamase to TEM-52 toward extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and noncovalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM β-lactamase antibiotic resistance.
酶活性位点静电作用与化学定位的相互作用对于理解酶催化的起源以及新型催化剂的设计至关重要。我们重建了TEM-1 β-内酰胺酶向TEM-52扩展光谱活性的进化轨迹，以更好地理解抗生素耐药性的出现，并深入了解催化过程中涉及的结构-功能范式和非共价相互作用。利用详细的动力学分析和振动斯塔克效应，我们量化了青霉素G和头孢噻肟在米氏复合物和酰基酶复合物中的速率和电场变化，以确定电场对调节功能的进化作用。这些数据与分子动力学模拟相结合，以解释和量化进化过程中底物依赖性的结构变化。我们观察到，这一进化轨迹利用了一个大型的预组织电场和底物依赖性化学定位来促进催化作用。这决定了TEM β-内酰胺酶抗生素耐药性中的进化能力、底物混杂性和蛋白质适应性格局。
