Fernandez A Z, Tablante A, Beguín S, Hemker H C, Apitz-Castro R
Lab. Trombosis Experimental, Centro de Biofísica y Bioquímica, IVIC, Ap. 21827, Caracas, Venezuela.
Biochim Biophys Acta. 1999 Sep 14;1434(1):135-42. doi: 10.1016/s0167-4838(99)00160-0.
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.
已确定水蛭素对活化因子X（FXa）的动力学作用机制。水蛭素在接近等摩尔浓度（摩尔比为2 - 6倍）孵育数秒内即可抑制活化因子X。将数据拟合至紧密结合抑制剂方程得出K(i)(K(d)) = 14.8±1.5 nM。水蛭素 - FXa复合物的形成可通过两步机制来解释，第一步为可逆步骤，k(on) = 1.117（±0.169，标准误）×10⁶ M⁻¹s⁻¹，k(off) = 15.388（±1.672）×10⁻³ s⁻¹；第二步为不可逆步骤，与浓度无关，k(2) = 0.072 s⁻¹。由k(off)/k(on)得出的K(d)为13.76 nM。Lineweaver - Burk图显示为非竞争性行为。水蛭素在完全抑制浓度下不影响对氨基苯甲脒与FXa的结合，这一观察结果支持了水蛭素的这种非竞争性抑制模式。此外，在相同条件下，水蛭素使对氨基苯甲脒 - FXa复合物的荧光强度降低不到14%。我们得出结论，水蛭素是FXa的非竞争性、紧密结合抑制剂，这一特性在天然FXa抑制剂中迄今为止是独一无二的。
