Wolberg A S, Morris D P, Stafford D W
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Biochemistry. 1997 Apr 8;36(14):4074-9. doi: 10.1021/bi962274y.
Factor IX activation by factor XIa is thought to proceed through the singly-cleaved free intermediate, factor IX alpha. However, we observed no intermediate development during factor IX activation by factor XIa when using a low substrate to enzyme ratio (44:1 mol/mol). This result can be explained by one of two mechanisms: (1) factor XIa-catalyzed activation proceeds via a singly-cleaved free intermediate with a much higher efficiency of cleavage than factor IX zymogen, or (2) the reaction occurs without free intermediate generation, whereby factor XIa makes both proteolytic cleavages in a single substrate molecule before releasing the final product (processive mechanism). We compared the factor XIa cleavage rates of free factor IX alpha and factor IXa alpha with that of factor IX zymogen. In contrast to the requirements of mechanism (1), the cleavage rate constants of factor IX zymogen, factor IX alpha, and factor IXa alpha were similar: 0.38 +/- 0.02 s(-1), 0.34 +/- 0.05 s(-1), and 0.27 +/- 0.01 s(-1), respectively. It seems likely that factor XIa-generated intermediates observed under some reaction conditions are produced through the occasional failure of a processive mechanism. Indeed, in reactions using a high substrate to enzyme ratio (1900:1 mol/mol), we observed some factor IX alpha development; however, the pattern of intermediate and product development over time was inconsistent with a mechanism involving an obligate intermediate. Rather, it corresponded to behavior expected from a processive mechanism undergoing a consistent low failure. We conclude that factor XIa-catalyzed activation of factor IX proceeds via a processive mechanism without release of a free intermediate.
因子 XIa 激活因子 IX 被认为是通过单链裂解的游离中间体——因子 IXα 进行的。然而，当使用低底物与酶的比例（44:1 摩尔/摩尔）时，我们在因子 XIa 激活因子 IX 的过程中未观察到中间体的形成。这一结果可以用两种机制之一来解释：（1）因子 XIa 催化的激活过程通过单链裂解的游离中间体进行，其裂解效率比因子 IX 酶原高得多；或者（2）反应发生时不产生游离中间体，即因子 XIa 在释放最终产物之前在单个底物分子中进行两次蛋白水解裂解（连续机制）。我们比较了游离因子 IXα、因子 IXaα 和因子 IX 酶原的因子 XIa 裂解速率。与机制（1）的要求相反，因子 IX 酶原、因子 IXα 和因子 IXaα 的裂解速率常数相似：分别为 0.38±0.02 s⁻¹、0.34±0.05 s⁻¹ 和 0.27±0.01 s⁻¹。在某些反应条件下观察到的因子 XIa 产生的中间体似乎可能是由于连续机制偶尔失败而产生的。事实上，在使用高底物与酶的比例（1900:1 摩尔/摩尔）的反应中，我们观察到了一些因子 IXα 的形成；然而，中间体和产物随时间的发展模式与涉及必需中间体的机制不一致。相反，它与连续机制经历一致的低失败率所预期的行为相符。我们得出结论，因子 XIa 催化的因子 IX 激活通过连续机制进行，不释放游离中间体。
Zotero 插件