title: Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.
abstract: The reaction pathway for inhibition of human factor Xa (fXa) by recombinant tick anticoagulant peptide (rTAP) was studied by stopped-flow fluorometry. In the presence of the fluorogenic substrate N-tert-butyloxycarbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl-7-amido-4 - methylcoumarin (B-IEGR-AMC) and under pseudo-first-order conditions, inhibition appears to occur via a two-step process. Initially, a weak enzyme-inhibitor complex forms with a dissociation constant (Ki) of 68 +/- 6 microM. The initial complex then rearranges to a more stable fXa-rTAP complex with a rate constant (k2) of 123 +/- 5 s-1. The apparent second-order rate constant (k2/Ki) describing formation of the stable complex is (1.8 +/- 0.2) x 10(6) M-1 s-1. Studies of the reaction of rTAP with fXa in the presence of the fluorescent active-site probe p-amino-benzamidine (P) revealed a reaction pathway wherein rTAP initially binds to the fXa-P complex in a two-step process prior to displacing P from the active site. These results indicate that rTAP can bind fXa via a site distinct from the active site (an exosite). The subsequent displacement of P from the active site of fXa by rTAP exhibits a dependence on the concentration of P, indicating that rTAP is locked into the active site in a third step.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Biological Chemistry Department, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: S P
authors.initials: SP
authors.lastname: Jordan
authors.forename: S S
authors.initials: SS
authors.lastname: Mao
authors.forename: S D
authors.initials: SD
authors.lastname: Lewis
authors.forename: J A
authors.initials: JA
authors.lastname: Shafer
mesh_terms: D000818:Animals; D060829:Arthropod Proteins; D001550:Benzamidines / Q000378:metabolism; D001665:Binding Sites; D015951:Factor Xa / Q000378:metabolism; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors; D013987:Ticks
chemical_list: D060829:Arthropod Proteins; D001550:Benzamidines; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D006629:Hirudins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C063967:tick anticoagulant peptide; C006500:4-aminobenzamidine; D013917:Thrombin; D015951:Factor Xa