Reactive site mutants of recombinant protein C inhibitor.
Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, alpha 1-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353-->P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.
Cooper
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Department of Pathology, University of North Carolina School of Medicine, Chapel Hill 27599-7035.
Church
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D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D018076:DNA, Complementary / Q000737:chemistry; D065427:Factor Xa Inhibitors; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000037:antagonists & inhibitors; D018046:Protein C Inhibitor / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D018076:DNA, Complementary; D065427:Factor Xa Inhibitors; D011486:Protein C; D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D013917:Thrombin