Barhoover Melissa A, Orban Tivadar, Beck Daniel O, Bukys Michael A, Kalafatis Michael
Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, USA.
Biochemistry. 2008 Jul 1;47(26):6840-50. doi: 10.1021/bi800057r. Epub 2008 Jun 7.
We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.
我们已经证明，凝血因子Va重链中的氨基酸E（323）、Y（324）、E（330）和V（331）是该辅因子与凝血因子Xa相互作用以及凝血酶原裂解最佳速率所必需的。我们还表明，氨基酸区域332 - 336包含对辅因子功能很重要的残基。使用重叠肽，我们确定氨基酸D（334）和Y（335）是辅因子活性的贡献者。我们构建了具有D（334）→K和Y（335）→F突变的重组凝血因子V分子（凝血因子V（KF））以及D（334）→A和Y（335）→A突变的重组凝血因子V分子（凝血因子V（AA））。动力学研究表明，虽然凝血因子Va（KF）和凝血因子Va（AA）对凝血因子Xa的解离常数（KD）与野生型凝血因子Va（凝血因子Va（WT））观察到的KD相似，但突变分子的凝血活性受损，并且与凝血因子Va（KF）和凝血因子Va（AA）组装的凝血酶原酶的催化常数（kcat）降低。与凝血因子Va（KF）或凝血因子Va（AA）组装的凝血酶原酶激活凝血酶原的二级速率常数比与凝血因子Va（WT）组装的凝血酶原酶催化相同反应的二级速率常数低约10倍。我们还创建了四重突变体，将氨基酸区域334 - 335中的突变与先前确定的对凝血因子Xa结合很重要的氨基酸（即E（323）Y（324）和E（330）V（331））的突变相结合。与四重突变体分子组装的凝血酶原酶显示的二级速率常数比与凝血因子Va（WT）组装的凝血酶原酶获得的值低高达400倍。数据表明，氨基酸区域334 - 335是凝血酶原酶有效催化凝血酶原所需的酶和底物重排所必需的。
