Document Type

Article

Language

eng

Format of Original

2 p.

Publication Date

10-2006

Publisher

American Chemical Society

Source Publication

Journal of the American Chemical Society

Source ISSN

0002-7863

Original Item ID

doi: 10.1021/ja0627343

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 (ε550 = 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.

Comments

Accepted version. Journal of the American Chemical Society, Vol. 128, No. 40 (October 2006): 13050-13051. DOI. © 2006 American Chemical Society. Used with permission.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

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