Department of Chemistry, York University, Toronto, Canada M3J 1P3.
Department of Chemistry, York University, Toronto, Canada M3J 1P3; Center for Research in Mass Spectrometry, York University, Toronto, Canada M3J 1P3.
J Mol Biol. 2018 Sep 14;430(18 Pt B):3311-3322. doi: 10.1016/j.jmb.2018.06.045. Epub 2018 Jun 28.
Conformational dynamics are increasingly recognized as being essential for enzyme function. However, there is virtually no direct experimental evidence to support the notion that individual dynamic modes are required for specific catalytic processes, apart from the initial step of substrate binding. In this work, we use a unique approach based on millisecond hydrogen-deuterium exchange mass spectrometry to identify dynamic modes linked to individual catalytic processes in the antibiotic resistance enzyme TEM-1 β-lactamase. Using a "good" substrate (ampicillin), a poorly hydrolyzed substrate (cephalexin) and a covalent inhibitor (clavulanate), we are able to isolate dynamic modes that are specifically linked to substrate binding, productive lactam ring hydrolysis and deacylation. These discoveries are ultimately translated into specific targets for allosteric TEM-1 inhibitor development.
构象动态变化越来越被认为是酶功能的关键。然而，除了底物结合的初始步骤之外，实际上几乎没有直接的实验证据支持这样一种观点，即特定的催化过程需要特定的动态模式。在这项工作中，我们使用一种基于毫秒氢氘交换质谱的独特方法，来鉴定与抗生素耐药酶 TEM-1 β-内酰胺酶中单个催化过程相关的动态模式。我们使用“良好”的底物（氨苄西林）、水解较差的底物（头孢氨苄）和共价抑制剂（克拉维酸），能够分离出与底物结合、产生活性内酰胺环水解和脱酰基作用特异性相关的动态模式。这些发现最终转化为变构 TEM-1 抑制剂开发的具体靶点。
