Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor I

• Published in: The Journal of biological chemistry Vol. 266; no. 13; pp. 8495 - 8500
• Main Authors: York, J D, Li, P, Gardell, S J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-05-1991
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Bacteriophages - genetics; Base Sequence; Binding Sites; Biological and medical sciences; DNA; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Molecular Sequence Data; Mutagenesis; Plasminogen Activators - antagonists & inhibitors; Plasminogen Inactivators - metabolism; Urokinase-Type Plasminogen Activator - antagonists & inhibitors; Human; Urokinase; Structure activity relation; Enzyme; Active site; Site directed mutagenesis; Enzyme inhibitor; Plasminogen activator; Inhibition; Ultraviolet visible spectrometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)93002-4

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.