title: Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism.
abstract: Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. rezaiear@slu.edu
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000235:genetics; D000925:Anticoagulants / Q000737:chemistry*; D001120:Arginine / Q000235:genetics; D060829:Arthropod Proteins; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002460:Cell Line; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D065427:Factor Xa Inhibitors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D026861:Ornithodoros / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D014443:Tyrosine / Q000235:genetics
chemical_list: D000925:Anticoagulants; D060829:Arthropod Proteins; D065427:Factor Xa Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide; D014443:Tyrosine; D010649:Phenylalanine; D015055:1-Carboxyglutamic Acid; D001120:Arginine; D015951:Factor Xa