Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of β-lactamase

Abstract

Nafcillin was shown to reversibly inhibit beta-lactamase from Staphylococcus aureus PC1 with characteristics indicative of a type A inhibitor [Citri, Samuni & Zyk (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052]. At nafcillin concentrations above 80 mM, complete inactivation occurred within 200 s. Upon removal of the excess nafcillin the inhibited enzyme was re-activated completely, with a rate constant of 2.0 x 10(-3) s-1 (25 degrees C). The inhibited enzyme was shown to be in the form of a covalent acyl-enzyme intermediate. Digestion by pepsin and trypsin yielded a single nafcillin-labelled peptide fragment which was isolated, sequenced and shown to be: Ala-Tyr-Ala-Ser-Thr-Ser-Lys. This sequence corresponds to the region surrounding the active-site serine residue, Ser-70, indicating that the inhibitor is covalently attached to the same residue as productive substrates.