Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.
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)
Jordan
S P
SP
Biological Chemistry Department, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
Mao
S S
SS
Lewis
S D
SD
Shafer
J A
JA
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
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