Extended-spectrum β-lactamases (ESBLs) of different Enterobacteriaceae (e.g. <i>Escherichia coli, Klebesiella pneumoniae</i>) have developed significant resistance mechanisms against antibiotics. ESBLs, like class A CTX-M enzymes hydrolyze the β-lactam ring of different antibiotics such as penicillins and cephalosporins. The hydrolysis reaction involves the formation of an acyl-enzyme complex by the CTX-M enzymes. The active site residues in the complex play a pivotal role in the catalysis. It is important to track the protonation state of catalytic residues to fully understand the mechanism behind this acylation-deacylation process. Here, we present NMR evidence showing that binding of a non-β-lactam ring containing antibiotic, avibactam, results in a significantly lower pKa of the catalytic lysine (K73) of CTX-M9 compared to that expected for free lysine. This altered pKa provides novel, important mechanistic understanding about the catalytic properties of these enzymes and thus their inhibition. K73 with lower pKa compared to the acyl-enzyme complex with β-lactams hinders the deprotonation of E166, which also ceases the initiation of the deacylation step.

NMR study of the proton transfer mechanism in class A β-lactamase catalysis and inhibition by avibactam

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.