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具有受限β-链构象的凝血因子VIIa二硫键锁定变体具有增强的酶活性。

Disulfide locked variants of factor VIIa with a restricted beta-strand conformation have enhanced enzymatic activity.

作者信息

Maun Henry R, Eigenbrot Charles, Raab Helga, Arnott David, Phu Lilian, Bullens Sherron, Lazarus Robert A

机构信息

Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

出版信息

Protein Sci. 2005 May;14(5):1171-80. doi: 10.1110/ps.041097505.

Abstract

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

摘要

将酶原因子VII蛋白水解加工为因子VIIa(FVIIa)是实现最大蛋白水解活性所必需的,但并不充分,这还需要在与辅因子组织因子(TF)结合时诱导产生的额外变构影响。影响FVIIa酶原性的一个关键构象变化涉及β链B2在其酶原形式和蛋白酶形式中的位置发生独特的三个残基位移。通过选择性地引入新的二硫键,我们将这些链的构象锁定为类似活性TF*FVIIa的状态。命名为136:160、137:159、138:160和139:157的FVIIa突变体反映了新二硫键的位置(胰凝乳蛋白酶原编号),通过TF亲和层析进行表达和纯化。对FVIIa突变体的胰蛋白酶肽段进行质谱分析,证实了新二硫键的形成。酰胺水解活性的动力学分析表明,与野生型相比,所有单独的FVIIa变体的比活性均有所增加,对于底物S-2765,最大的是136:160和138:160变体,分别增加了670倍和330倍。值得注意的是,FVIIa二硫键锁定变体在酰胺水解测定中不再需要TF作为最大活性的辅因子。在可溶性TF存在下,与野生型相比,136:160和138:160变体的活性分别提高了20倍和12倍。对于重新脂质化的TF,136:160和137:159突变体在激活FX时的V(max)/K(m)值也增加了约三倍,但在TF依赖性凝血测定中没有显著改善。因此,虽然在活性位点结合的酰胺水解底物获得了大幅的速率增强,但与FVIIa外位点结合的大分子底物需要更复杂的催化要求。

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