FACTOR-IX ACTIVATION BY FACTOR XIA PROCEEDS WITHOUT RELEASE OF A FREEINTERMEDIATE

Factor IX activation by facto; XIa is thought to proceed through the s ingly-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 resul t 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 reac tion occurs without free intermediate generation, whereby factor XIa m akes both proteolytic cleavages in a single substrate molecule before releasing the final product (processive mechanism). We compared the fa ctor XIa cleavage rates of free factor IX alpha and factor IXa alpha w ith that of factor IX zymogen. In contrast to the requirements of mech anism (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 c onditions 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; howev er, the pattern of intermediate and product development over time was inconsistent with a mechanism involving an obligate intermediate. Rath er, it corresponded to behavior expected from a processive mechanism u ndergoing a consistent low failure. We conclude that factor XIa-cataly zed activation of factor IX proceeds via a processive mechanism withou t release of a free intermediate.