title: 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.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, Ohio 45056, USA.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Matthews
authors.forename: Gopalraj
authors.initials: G
authors.lastname: Periyannan
authors.forename: Christine
authors.initials: C
authors.lastname: Hajdin
authors.forename: Tara K
authors.initials: TK
authors.lastname: Sidgel
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D003035:Cobalt; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase