title: Conversion of the substrate specificity of mouse proteinase granzyme B.
abstract: Mouse granzyme B is the prototypic member of a subfamily of serine proteinases expressed in cytolytic lymphocytes. Molecular modelling of granzyme B indicated that the side chain of Arg 208 partially fills the specificity pocket, thus predicting the preference of this enzyme for substrates containing acidic side chains, a feature unique among eukaryotic serine proteinases. Replacement of Arg 208 with glycine results in an enzyme lacking this activity, but which is able to hydrolyze hydrophobic substrates. These results demonstrate unequivocally that the substrate preference of granzyme B is determined by a positive charge in the specificity pocket and also represent one of the few examples of rational and efficient alteration of serine proteinase substrate-specificity following a single amino acid substitution.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Caputo
authors.forename: M N
authors.initials: MN
authors.lastname: James
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
authors.forename: D
authors.initials: D
authors.lastname: Hudig
authors.forename: R C
authors.initials: RC
authors.lastname: Bleackley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001593:Benzyl Compounds / Q000378:metabolism; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D005998:Glycine / Q000737:chemistry; D053804:Granzymes; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D001593:Benzyl Compounds; D011993:Recombinant Fusion Proteins; D001120:Arginine; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; D005998:Glycine