title: Comparative studies on P2 specificity of wild-type rat tissue kallikrein, Y99H:W215G mutant and tonin.
abstract: To probe residues responsible for P2 specificity, we have recently created a mutant enzyme from rat tissue kallikrein with Tyr99 to His and Trp215 to Gly exchange (Y99H:W215G) using site-specific mutagenesis. In the present study, additional characterization of substrate specificities of both wild-type tissue kallikrein, Y99H:W215G mutant and native tonin was performed using synthetic Ac-X-Arg-pNA substrates especially designed for testing P2 specificity. Kinetic analyses of Km and kcat demonstrate a clear correlation between dramatically reduced affinity for hydrophobic P2 side-chain and the loss of the Tyr99-Trp215 hydrophobic pair. Analyses of rat tonin reveal a correlation between increased reaction rate and P2 hydrophilicity although tonin displays similar pattern in P2 affinity as compared with tissue kallikrein, suggesting a less hydrophobic environment in the substrate-binding pocket of rat tonin. The results strongly support the hypothesis that Tyr99-Trp215 interaction is the major determinant for P2 specificity and that the presence of a hydrophobic side-chain in P2 position substantially facilitates substrate hydrolysis of tissue kallikrein-like enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Chao
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: L
authors.initials: L
authors.lastname: Chao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004151:Dipeptides; D006801:Humans; D007610:Kallikreins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
chemical_list: D004151:Dipeptides; D007703:Peptidyl-Dipeptidase A; D007610:Kallikreins