Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of beta-lactamase.
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.
Tan
A K
AK
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Fink
A L
AL
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D007700:Kinetics; D008962:Models, Theoretical; D009254:Nafcillin / Q000378:metabolism* / Q000494:pharmacology; D010400:Penicillin G / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
D010446:Peptide Fragments; D065093:beta-Lactamase Inhibitors; D009254:Nafcillin; D001618:beta-Lactamases; D010400:Penicillin G