Bharadwaj D, Iino M, Kontoyianni M, Smith K J, Foster D C, Kisiel W
Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
J Biol Chem. 1996 Nov 29;271(48):30685-91. doi: 10.1074/jbc.271.48.30685.
Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.
因子VII是一种维生素K依赖的丝氨酸蛋白酶原，参与血液凝固的起始阶段。在一名患有严重因子VII缺乏症的24岁男性中鉴定出一种因子VII分子变异体（因子VII Central），其血浆因子VII抗原为正常的38%，但因子VII促凝活性表达<1%。对该患者因子VII基因的DNA序列分析显示，外显子VIII中核苷酸10907处胸腺嘧啶向胞嘧啶的转变导致了Phe328到Ser的新氨基酸替代。该患者对此突变是纯合子，而患者的每个父母对此突变是杂合子。为了研究该变异体的分子特性，制备了重组F328S因子VII突变体并与野生型因子VII进行相关分析。F328S因子VII表现出<1%的因子VII促凝活性，对组织因子的亲和力降低了2倍，并且在被因子Xa激活后，在组织因子存在的情况下无法激活因子X或因子IX。此外，F328S因子VIIa在组织因子存在下未表现出可检测到的酰胺水解活性。如SDS凝胶的光密度扫描所示，相对于野生型因子VII的激活速率，F328S因子VII被因子Xa激活的速率明显降低。通过SDS-聚丙烯酰胺凝胶电泳对该反应进行时间分析还显示，完整的F328S因子VII中Arg315-Lys316肽键被因子Xa蛋白水解后形成了两种新的F328S因子VII降解产物（40和9 kDa）。因子VIIa丝氨酸蛋白酶结构域的计算机建模和分子动力学模拟表明，F328S因子VIIa无法切割底物可能是由于Tyr377和Asp338之间明显形成了氢键，Asp338是底物结合口袋底部的一个残基，对底物精氨酸侧链与酶的相互作用很重要。这些发现表明，在凝血酶原、因子IX、因子X、因子VII和胰蛋白酶中保守的Phe328对因子VIIa催化很重要。
