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.
Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA.
Deley
D
Sulton
Doritza
D
Pagan-Rodriguez
Xiang
X
Zhou
Yiding
Y
Liu
Andrea M
AM
Hujer
Christopher R
CR
Bethel
Marion S
MS
Helfand
Jodi M
JM
Thomson
Vernon E
VE
Anderson
John D
JD
Buynak
Lily M
LM
Ng
Robert A
RA
Bonomo
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002853:Chromatography, Liquid; D019818:Clavulanic Acid / Q000494:pharmacology*; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D010455:Peptides; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D012694:Serine; D014357:Trypsin; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D005998:Glycine