Larsen Katrine S, Østergaard Henrik, Bjelke Jais R, Olsen Ole H, Rasmussen Hanne B, Christensen Leif, Kragelund Birthe B, Stennicke Henning R
Haemostasis Biochemistry, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
Biochem J. 2007 Aug 1;405(3):429-38. doi: 10.1042/BJ20061901.
The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several exosites have been identified, whereas only little is known about the specificity dictated by the active-site architecture. In the present study, we have profiled the primary P4-P1 substrate specificity of FVIIa using positional scanning substrate combinatorial libraries and evaluated the role of the selective active site in defining specificity. Being a trypsin-like serine protease, FVIIa had P1 specificity exclusively towards arginine and lysine residues. In the S2 pocket, threonine, leucine, phenylalanine and valine residues were the most preferred amino acids. Both S3 and S4 appeared to be rather promiscuous, however, with some preference for aromatic amino acids at both positions. Interestingly, a significant degree of interdependence between the S3 and S4 was observed and, as a consequence, the optimal substrate for FVIIa could not be derived directly from a subsite-directed specificity screen. To evaluate the role of the active-site residues in defining specificity, a series of mutants of FVIIa were prepared at position 239 (position 99 in chymotrypsin), which is considered to be one of the most important residues for determining P2 specificity of the trypsin family members. This was confirmed for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered separately.
凝血蛋白酶对大分子底物具有极高的特异性，这是由涉及催化凹槽和远端外位点的众多相互作用所决定的。对于通过外源性途径启动凝血的主要因子FVIIa [活化的FVII（凝血因子VII）]，已经鉴定出了几个外位点，而关于活性位点结构所决定的特异性却知之甚少。在本研究中，我们使用位置扫描底物组合文库分析了FVIIa的主要P4 - P1底物特异性，并评估了选择性活性位点在定义特异性中的作用。作为一种类胰蛋白酶丝氨酸蛋白酶，FVIIa对P1的特异性仅针对精氨酸和赖氨酸残基。在S2口袋中，苏氨酸、亮氨酸、苯丙氨酸和缬氨酸残基是最优选的氨基酸。然而，S3和S4似乎都相当宽泛，在这两个位置都对芳香族氨基酸有一定偏好。有趣的是，观察到S3和S4之间存在显著程度的相互依赖性，因此，FVIIa的最佳底物不能直接从亚位点导向的特异性筛选中得出。为了评估活性位点残基在定义特异性中的作用，在239位（胰凝乳蛋白酶中的99位）制备了一系列FVIIa突变体，该位点被认为是决定胰蛋白酶家族成员P2特异性的最重要残基之一。对于FVIIa，这一点通过主要底物特异性的显著变化和抗凝血酶III抑制率的降低得到了证实。有趣的是，这些变化不一定与激活因子X的能力改变相一致，这表明抑制剂和大分子底物的选择性可以分别进行设计。
