title: Distinct mechanisms contribute to stringent substrate specificity of tissue-type plasminogen activator.
abstract: Tissue-type plasminogen activator (t-PA) has evolved to optimize cleavage of plasminogen (Plg) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2 subsites of t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly selective cleavage of protein fusions. Surprisingly, t-PA cleaves this engineered protein substrate with a Km that is reduced 950-fold relative to the Km for hydrolysis of the same target sequence within a peptide. This reduction of Km suggests that binding is facilitated by interactions between protein substrate and protease that are distant from the P4-P2' residues. We use this kinetic data to derive a model in which several distinct mechanisms contribute to the remarkable specificity of t-PA.
authors.affiliation: Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 75235, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Coombs
authors.forename: A T
authors.initials: AT
authors.lastname: Dang
authors.forename: E L
authors.initials: EL
authors.lastname: Madison
authors.forename: D R
authors.initials: DR
authors.lastname: Corey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D010958:Plasminogen; D014357:Trypsin; D010959:Tissue Plasminogen Activator; D009955:Ornithine Decarboxylase