Clavulanic acid inactivation of SHV-1 and the inhibitor-resistant S130G SHV-1 beta-lactamase. Insights into the mechanism of inhibition.

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-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-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-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 S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-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 beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition.

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.