title: Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor I.
abstract: Plasminogen activator inhibitor (PAI-I) rapidly inactivates tissue plasminogen activator (t-PA) and urokinase (UK) with nearly identical association rate constants. The contributions of Ser344, Ala345, and Arg346 (P3, P2, and P1 residues, respectively) in PAI-I to inhibition of UK and t-PA were evaluated using combinatorial mutagenesis of the human PAI-I cDNA. A bacteriophage lambda expression library potentially encoding the 8000 unique PAI-I species were screened for inhibitory activity against UK using a fibrin indicator gel. 390 plaques demarcated by zones of retarded fibrinolysis were analyzed to determine the DNA sequences of their associated active PAI-1 species. We found 134 unique PAI-1 variants that retained inhibitory activity towards UK; they contained a variety of amino acids in their P3 and P2 positions but only Arg or, infrequently, Lys in their P1 position. Each of the unique active PAI-1 were assayed for inhibitory activity towards UK or t-PA; many substitutions differentially affected the ability of the inhibitor to inactivate UK and t-PA. For example, replacement of Ser344 and Ala344 with Val and Pro, respectively, yielded a PAI-1 variant exhibiting an association rate constant that was unchanged for t-PA but decreased 23-fold for UK, relative to native PAI-1. In general, the PAI-1 variants were more potent inhibitors of t-PA than UK. Hence, t-PA appears more tolerant than UK of structural diversity present in the P3 and P2 positions of the PAI-1 variants.
authors.affiliation: Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: J D
authors.initials: JD
authors.lastname: York
authors.forename: P
authors.initials: P
authors.lastname: Li
authors.forename: S J
authors.initials: SJ
authors.lastname: Gardell
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
chemical_list: D015849:Plasminogen Inactivators; D004247:DNA; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator