title: Internally quenched peptides for the study of lysostaphin: An antimicrobial protease that kills Staphylococcus aureus.
abstract: Lysostaphin (EC. 3.4.24.75) is a protein secreted by Staphylococcus simulans biovar staphylolyticus and has been shown to be active against methicillin resistant S. aureus (MRSA). The design and synthesis of three internally quenched substrates for lysostaphin based on the peptidoglycan crossbridges of S. aureus, and their use in fluorescence resonance energy transfer (FRET) assays is reported. These substrates enabled the gathering of information about the endopeptidase activity of lysostaphin and the effect that mutations have on its enzymatic ability. Significant problems with the inner filter effect and substrate aggregation were encountered; their minimisation and the subsequent estimation of the kinetic parameters for the interaction of lysostaphin with the substrates is described, as well as a comparison of substrates incorporating two FRET pairs: Abz-EDDnp and DABCYL-EDANS. In addition to this, the points of cleavage caused by lysostaphin in Abz-pentaglycine-EDDnp have been determined by HPLC and mass spectrometry analysis to be between glycines 2 and 3(approximately 60%) and glycines 3 and 4 (approximately 40%).
authors.affiliation: School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, UK NG7 2RD.
authors.forename: Rachel
authors.initials: R
authors.lastname: Warfield
authors.forename: Philip
authors.initials: P
authors.lastname: Bardelang
authors.forename: Helen
authors.initials: H
authors.lastname: Saunders
authors.forename: Weng C
authors.initials: WC
authors.lastname: Chan
authors.forename: Christopher
authors.initials: C
authors.lastname: Penfold
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Neil R
authors.initials: NR
authors.lastname: Thomas
mesh_terms: D005453:Fluorescence; D007700:Kinetics; D008248:Lysostaphin / Q000737:chemistry / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000737:chemistry*; D010457:Peptidoglycan / Q000737:chemistry; D013211:Staphylococcus aureus / Q000187:drug effects*; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D010455:Peptides; D010457:Peptidoglycan; D008248:Lysostaphin