title: The kinetic assembly of the intrinsic bovine factor X activation system.
abstract: The activation of bovine factor X by bovine factors IXa alpha and IXa beta has been examined under conditions of progressive assembly of the complete intrinsic activation system, i.e., factor X/factor IXa/Ca2+/phospholipid (PL)/factor VIIIa. In the presence of Ca2+, and the absence of PL and factor VIIIa, factor IXa alpha is a more efficient enzyme than factor IXa beta toward factor X activation, primarily due to the much higher kcat for the factor IXa alpha-catalyzed reaction. Analysis of the steady-state kinetic properties, after addition of PL (mixtures of phosphatidylcholine/phosphatidylserine) to the factor X/factor IXa/Ca2+ activation system, shows that the mechanism most closely follows a nonessential activation scheme, where the true substrate is the factor X/Ca2+/PL complex. The presence of PL results in a large (1-2 orders of magnitude) increase of the kcat for factor IXa beta, but does not substantially affect the steady-state kinetic constants of the factor IXa alpha-catalyzed reaction. Examination of the steady-state activation kinetics of factor X, after addition of factor VIIIa to factor X/factor IXa/Ca2+/PL, demonstrates that the mechanism is most consistent with a nonessential activation scheme of fluid phase substrate (factor X) being activated by a PL-bound enzyme system (factor IXa/Ca2+/factor VIIIa/PL). The presence of factor VIIIa stimulated the rates of factor X activation by factor IXa beta/Ca2+/PL by 1-2 orders of magnitude. Qualitatively similar behavior was noted for the factor IXa alpha-catalyzed activation. The results of this manuscript show that, in the presence of Ca2+ and absence of other cofactors, factor IXa alpha is a much more efficient enzyme for factor X activation, as compared to factor IXa beta. This is likely due to effects on the system resulting from covalent retention of the negatively charged activation peptide, by factor IXa alpha. However, the enzymatic activity of factor IXa beta shows a far better response to cofactors, particularly PL, than factor IXa alpha, thereby rendering factor IXa beta the more efficient enzyme in the complete intrinsic activation system.
authors.affiliation: Department of Chemistry, University of Notre Dame, Indiana 46556.
authors.forename: J M
authors.initials: JM
authors.lastname: Beals
authors.forename: B A
authors.initials: BA
authors.lastname: Chibber
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D005164:Factor IX / Q000378:metabolism; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D010743:Phospholipids / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D010743:Phospholipids; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D002118:Calcium