Clavulanic Acid Inactivation of SHV-1 and the Inhibitor-resistant S130G SHV-1 β-Lactamase: INSIGHTS INTO THE MECHANISM OF INHIBITION.

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1 β-lactamases, enzymes that confer resistance to β-1actams in many Gram-negative pathogens. This compound has enjoyed widespread clinical use as part of β-lactam β-1actamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 β-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser ↵ Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1 β-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant β-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 β-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130G β-lactamases (± inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gβ-1actanmses inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 mn are diminished and delayed when S130G β-1actamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G variant β-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant β-1actamases and suggest how strategic chemical design can lead to novel ways to inhibit β-lactamases. [ABSTRACT FROM AUTHOR]