Matthews Megan L, Periyannan Gopalraj, Hajdin Christine, Sidgel Tara K, Bennett Brian, Crowder Michael W
Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, Ohio 45056, USA.
J Am Chem Soc. 2006 Oct 11;128(40):13050-1. doi: 10.1021/ja0627343.
In an effort to probe the reaction mechanism of VanX, the d-ala-d-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (epsilon550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts.
为了探究VanX（细菌中高水平耐万古霉素所需的D-丙氨酰-D-丙氨酸二肽酶）的反应机制，对与D-丙氨酰-D-丙氨酸反应的钴（II）取代酶进行了停流动力学和速冻淬灭电子顺磁共振（EPR）研究。当VanX与底物反应时，550 nm处钴（II）配体场带的强度降低（ε550从140降至18 M-1 cm-1），这表明底物结合后金属的配位数从5增加到6。停流曲线拟合到一个动力学机制，该机制表明存在一个中间体，其分解是限速步骤。速冻淬灭EPR研究证实了存在一种反应中间体，其超精细常数异常低（33 G），这表明中间体与金属中心呈双齿配位。EPR研究还鉴定出一种独特的酶-产物复合物。这些结果被用于提供VanX的详细反应机制，可用于指导未来的抑制剂设计工作。
