TY - JOUR
T1 - Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X
AU - Fernandez, Ana Z.
AU - Tablante, Alfonso
AU - Beguin, Suzette
AU - Hemker, H. Coenraad
AU - Apitz-Castro, Rafael
PY - 1999/9/14
Y1 - 1999/9/14
N2 - The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K-i(K-d) = 14.8 +/- 1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M-1 s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K-d obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces < 14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.
AB - The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K-i(K-d) = 14.8 +/- 1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M-1 s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K-d obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces < 14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors. (C) 1999 Elsevier Science B.V. All rights reserved.
U2 - 10.1016/S0167-4838(99)00160-0
DO - 10.1016/S0167-4838(99)00160-0
M3 - Article
SN - 0167-4838
VL - 1434
SP - 135
EP - 142
JO - Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
IS - 1
ER -