1. Academic Validation
  2. Identification of the site of covalent attachment of nafcillin, a reversible suicide inhibitor of beta-lactamase

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

  • Biochem J. 1992 Jan 1;281 ( Pt 1)(Pt 1):191-6. doi: 10.1042/bj2810191.
A K Tan 1 A L Fink
Affiliations

Affiliation

  • 1 Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
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.

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