title: Delocalizing trypsin specificity with metal activation.
abstract: Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California at San Francisco 94143-0446, USA.
authors.forename: W S
authors.initials: WS
authors.lastname: Willett
authors.forename: L S
authors.initials: LS
authors.lastname: Brinen
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D007611:Aprotinin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D033903:Periplasmic Proteins; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D033903:Periplasmic Proteins; D014443:Tyrosine; D012694:Serine; D007611:Aprotinin; D014357:Trypsin