Structure-function studies of the SERPIN plasminogen activator inhibitor type 1. Analysis of chimeric strained loop mutants.
Three chimeric mutants of plasminogen activator inhibitor 1 (PAI-1) have been constructed where the strained loop of wild type PAI-1 (wtPAI-1) has been replaced with a 19-amino acid region from either plasminogen activator inhibitor 2 (PAI-2), antithrombin III, or with an artificial serine protease inhibitor superfamily consensus strained loop. The inhibitors were expressed in Escherichia coli, and the purified proteins had specific activities toward urokinase-type plasminogen activator (uPA) or the single- and two-chain forms of tissue type plasminogen activator (tPA) that were similar to wtPAI-1. Experiments suggest that the strained loop of PAI-1 is not responsible for the transition between the latent and the active conformations or for binding to vitronectin. Second-order rate constants for the interactions with uPA and single- or two-chain tPA were similar to those of wtPAI-1. Values range from a low of 1.8 x 10(5) M-1 s-1 for the interaction of the PAI-2 chimera with single-chain tPA to a high value of 1.6 x 10(7) M-1 s-1 for the consensus mutant with two-chain tPA. This former value is 200 times higher than the reported rate constant for the interaction between PAI-2 and single-chain tPA, suggesting that structures outside of the strained loop are responsible for the major differences in specificity between PAI-1 and PAI-2.
Department of Applied Cell and Molecular Biology, University of Umeå, Sweden.
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Lawrence
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Strandberg
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Ericson
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D019096:Vitronectin
D006023:Glycoproteins; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators; D019096:Vitronectin