Probing the non-proline Cis peptide bond in beta-lactamase from Staphylococcus aureus PC1 by the replacement Asn136-Ala

The presence of a non-proline cis peptide between Glu166 and Ile 167 in the active site of beta-lactamase from Staphylococcus aureaus PC1 was determined. A site-directed mutant N136A was produced to examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166. No measurable hydrolytic activity towards penicillins or cephalosporis by the enzyme was observed, apart from the chromegenic cephalosporin, nitrocefin. The hydrolysis of nitrocefin after reincubation with several beta-lactam compounds was monitored, and cephalosporins were found to form stable acyl complexes with the enzyme.Electrospray mass spectrometry was used to determine the molecular weight of the mutant.