Abstract

β-lactamases hydrolyze penicillin, cephalosporin, and carbapenemβ-lactam antibiotics. The class D β-lactamase OXA-1 has a catalytic serine (position 67) thought to be deprotonated and thereby activated by an unusual N^ζ-carboxylated lysine (position 70). We have made several mutations of OXA-1 at both K70 and S67 to help elucidate the role of these two critical residues in the catalytic mechanism. We have used the fluorescent substrate BOCILLIN FL to demonstrate that the K70 mutants can acylate but are severely impaired for deacylation of various substrates. Interestingly, deacylation rates vary depending on the identity of the substituting residue, from t_1/2 = 40 min for K70A to undetectable deacylation for K70D. We have used tryptophan fluorescence spectroscopy to confirm that these results are applicable to natural (i.e. non-fluorescent) substrates.