title: Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site.
abstract: Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.
authors.affiliation: Department of Chemistry and Biochemistry, Molecular Biology Institute and UCLA-Department of Energy Institute for Genomics and Proteomics, UCLA, Los Angeles, California 90095-1570, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Connolly
authors.forename: Brenton T
authors.initials: BT
authors.lastname: Smith
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Pilpa
authors.forename: Udayar
authors.initials: U
authors.lastname: Ilangovan
authors.forename: Michael E
authors.initials: ME
authors.lastname: Jung
authors.forename: Robert T
authors.initials: RT
authors.lastname: Clubb
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000502:physiology*; D001426:Bacterial Proteins; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry*; D007477:Ions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010458:Peptidyl Transferases / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013450:Sulfones / Q000037:antagonists & inhibitors; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D007093:Imidazoles; D007477:Ions; D010455:Peptides; D013438:Sulfhydryl Compounds; D013450:Sulfones; D006639:Histidine; C009873:divinyl sulfone; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D003546:Cysteine Endopeptidases; D003545:Cysteine