Tethered processivity of the vitamin K-dependent carboxylase: Factor IX isefficiently modified in a mechanism which distinguishes Gla's from Glu's and which accounts for comprehensive carboxylation in vivo

The vitamin K-dependent (VKD) carboxylase binds VKD proteins via their prop eptide and converts Glu's to gamma -carboxylated Glu's, or Gla's, in the Gl a domain. Multiple carboxylation is required for activity, which could be a chieved if the carboxylase is processive. In the only previous study to tes t for this capability, an indirect assay was used which suggested processiv ity; however, the efficiency was poor and raised questions regarding how fu ll carboxylation is accomplished. To unequivocally determine if the carboxy lase is processive and if it can account for comprehensive carboxylation in vivo, as well as to elucidate the enzyme mechanism, we developed a direct test for processivity. The in vitro carboxylation of a complex containing c arboxylase and full-length factor IX (fIX) was challenged with an excess am ount of a distinguishable fIX variant. Remarkably, carboxylation of fIX in the complex was completely unaffected by the challenge protein, and compreh ensive carboxylation was achieved, showing conclusively that the carboxylas e is processive and highly efficient. These studies also showed that carbox ylation of individual fIX/carboxylase complexes was nonsynchronous and impl icated a driving force for the reaction which requires the carboxylase to d istinguish Glu's from Gla's. We found that the Gla domain is tightly associ ated with the carboxylase during carboxylation, blocking the access of a sm all peptide substrate (EEL). The studies describe the first analysis of pre formed complexes, and the rate for full-length, native fIX in the complex w as equivalent to that of the substrate EEL. Thus, intramolecular movement w ithin the Gla domain to reposition new Glu's for catalysis is as rapid as d iffusion-limited positioning of a small substrate, and the Gla domain is no t sterically constrained by the rest of the fIX molecule during carboxylati on. The rate of carboxylation of fIX in the preformed complex was 24-fold h igher than for fIX modified by free carboxylase, which supports carboxylase processivity and which indicates that binding and/or release is the rate-l imiting step in protein carboxylation. These data indicate a model of tethe red processivity, in which the VKD proteins remain bound to the carboxylase throughout the reaction via their propeptide, while the Gla domain undergo es intramolecular movement to reposition new Glu's for catalysis to ultimat ely achieve comprehensive carboxylation.