title: Electrostatic reversal of serine proteinase substrate specificity.
abstract: Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Caputo
authors.forename: J C
authors.initials: JC
authors.lastname: Parrish
authors.forename: M N
authors.initials: MN
authors.lastname: James
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
authors.forename: R C
authors.initials: RC
authors.lastname: Bleackley
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D053804:Granzymes; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases