Mutation of the active site carboxy-lysine of OXA-1 β-lactamase results in deacylation-deficient enzyme
β-lactamases hydrolyze penicillin, cephalosporin and carbapenem β-lactam antibiotics. The class D β-lactamase OXA-1 has a catalytic serine (position 67) thought to be deprotonated and thereby activated by a carboxylated lysine (position 70). We have made several mutations of OXA-1 at both Lys70 and Ser67 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 Lys70 mutant can acylate but is severely impaired for deacylation of various substrates. Interestingly, deacylation rates vary depending on the identity of substituting residue, from t1/2 = 30 min for Lys70Ala to undetectable deacylation for Lys70Asp. We have used tryptophan fluorescence spectroscopy to confirm that these results are applicable to natural (i.e. Non-fluorescent) substrates.
Faculty Mentor: David Leonard
Kyle presented at the Annual American Society of Biochemistry and Molecular Biology April 17-21, 2009 in New Orleans, LA.
Page last modified January 21, 2011