Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase.

ABSTRACT: Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuat

SUBMITTER: Raber ML 

PROVIDER: S-EPMC2610506 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature