Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor I.
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.
Duke Scholars
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Related Subject Headings
- Urokinase-Type Plasminogen Activator
- Plasminogen Inactivators
- Plasminogen Activators
- Mutagenesis
- Molecular Sequence Data
- Humans
- DNA
- Biochemistry & Molecular Biology
- Binding Sites
- Base Sequence
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Urokinase-Type Plasminogen Activator
- Plasminogen Inactivators
- Plasminogen Activators
- Mutagenesis
- Molecular Sequence Data
- Humans
- DNA
- Biochemistry & Molecular Biology
- Binding Sites
- Base Sequence