Kotkow K J, Deitcher S R, Furie B, Furie B C
Division of Hematology-Oncology, New England Medical Center, Boston, Massachusetts.
J Biol Chem. 1995 Mar 3;270(9):4551-7. doi: 10.1074/jbc.270.9.4551.
The incorporation of factor Xa into the prothrombinase complex, factor Xa-factor Va-phospholipid-Ca(II), results in an approximately 10(5)-fold higher rate of substrate activation than that of the enzyme alone. To examine the role of the prothrombin kringle domains in the interaction with prothrombinase we have employed site-directed mutagenesis to produce prothrombin species that lack either the first kringle domain, PT/delta K1, or the second kringle domain, PT/delta K2. Previously, we have shown that these proteins are fully carboxylated and that they bind to phospholipid vesicles. In this investigation we demonstrate that cleavage at Arg271-Thr272 and Arg320-Ile321 peptide bonds occurs upon activation with prothrombinase to yield normal thrombin from both PT/delta K1 and PT/delta K2. In the absence of factor Va, the Km(app) for the activation of PT/delta K1, PT/delta K2, or plasma-derived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent. The Km(app) for the activation of PT/delta K2 by prothrombinase is approximately 4-5-fold higher than that obtained for plasma-derived prothrombin or PT/delta K1. These data demonstrate that the prothrombin kringle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction. In the absence of factor Va, equivalent kcat values were obtained for all of the prothrombin species when they were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold lower kcat value was obtained for the activation of PT/delta K2 by prothrombinase as compared with that obtained for plasma prothrombin or PT/delta K1. Collectively, these data suggest that determinants within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of substrate turnover. Indeed, in contrast to plasma-derived prothrombin, no direct binding of PT/delta K2 to factor Va to form the PT/delta K2-factor Va complex could be demonstrated by 90 degrees light scattering.
凝血因子Xa掺入凝血酶原酶复合物（凝血因子Xa - 凝血因子Va - 磷脂 - 钙离子(II)）后，底物激活速率比单独的酶高出约10^5倍。为了研究凝血酶原kringle结构域在与凝血酶原酶相互作用中的作用，我们采用定点诱变技术制备了缺乏第一个kringle结构域（PT/ΔK1）或第二个kringle结构域（PT/ΔK2）的凝血酶原变体。此前，我们已表明这些蛋白质已完全羧化且能与磷脂囊泡结合。在本研究中，我们证明在用凝血酶原酶激活时，PT/ΔK1和PT/ΔK2在精氨酸271 - 苏氨酸272和精氨酸320 - 异亮氨酸321肽键处发生裂解，产生正常的凝血酶。在缺乏凝血因子Va的情况下，凝血因子Xa - 磷脂 - 钙离子(II)激活PT/ΔK1、PT/ΔK2或血浆来源的凝血酶原的表观米氏常数（Km(app)）是相同的。凝血酶原酶激活PT/ΔK2的表观米氏常数比血浆来源的凝血酶原或PT/ΔK1高约4 - 5倍。这些数据表明，凝血酶原kringle结构域对底物 - 酶相互作用的结合亲和力没有显著贡献。在缺乏凝血因子Va的情况下，当所有凝血酶原变体被凝血因子Xa - 钙离子(II) - 磷脂激活时，获得了相同的催化常数（kcat）值。相比之下，与血浆凝血酶原或PT/ΔK1相比，凝血酶原酶激活PT/ΔK2的催化常数低7倍。总体而言，这些数据表明凝血酶原第二个kringle结构域内的决定簇与凝血因子Va相互作用，从而显著加速底物周转的催化速率。实际上，与血浆来源的凝血酶原不同，通过90度光散射无法证明PT/ΔK2与凝血因子Va直接结合形成PT/ΔK2 - 凝血因子Va复合物。
