Higashi S, Nishimura H, Fujii S, Takada K, Iwanaga S
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1992 Sep 5;267(25):17990-6.
We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.
我们设计了一种简单而灵敏的方法来测定因子VIIa - 组织因子复合物的活性，该方法使用Nα - 苄氧羰基 - L - 精氨酸对硝基苄酯（Z - Arg - ONb）作为底物（Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203 - 2209）。其原理是使用反相高效液相色谱法测量酯水解过程中释放的对硝基苄醇的量。Z - Arg - ONb对血浆丝氨酸蛋白酶和因子IXa具有广泛的特异性。使用该方法，我们研究了在各种条件下组织因子对因子VIIa酯酶活性的影响。我们发现组织因子极大地增强了因子VIIa催化的Z - Arg - ONb水解。即使存在组织因子，磷脂对于因子VIIa催化的Z - Arg - ONb水解也不是必需的。单独的因子VIIa对酯底物的Km值比对VIIa - 组织因子复合物的Km值高六倍（3.2对0.54 mM），而kcat值比对VIIa - 组织因子复合物的kcat值低12倍（14.3对173 s-1）。因此，组织因子显然影响因子VIIa的催化位点并增强酯底物的水解。当从因子VIIa中去除γ - 羧基谷氨酸结构域时，组织因子的这种增强作用消失，而在不存在组织因子的情况下酯酶活性不受此修饰的影响。即使反应混合物中不存在磷脂，γ - 羧基谷氨酸结构域可能也是与组织因子相互作用的有效决定因素所必需的。
