Probing the reaction mechanism of the D-ala-D-ala dipeptidase, VanX, by using stopped-flow kinetic and rapid-freeze quench EPR studies on the Co(II)-substituted enzyme. J Am Chem Soc 2006 Oct 11;128(40):13050-1
Date
10/05/2006Pubmed ID
17017774Pubmed Central ID
PMC2547881DOI
10.1021/ja0627343Scopus ID
2-s2.0-33749538082 (requires institutional sign-in at Scopus site) 16 CitationsAbstract
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.
Author List
Matthews ML, Periyannan G, Hajdin C, Sidgel TK, Bennett B, Crowder MWAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
Bacterial ProteinsCobalt
Electron Spin Resonance Spectroscopy
Kinetics
Metalloproteins
Serine-Type D-Ala-D-Ala Carboxypeptidase