Effects of Water Soluble Phosphotidylserine on Bovine Factor X a: Functional and Structural Changes Plus Dimerization

Abstract

Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X a. Activity measurements along with the fluorescence of active-site-labeled human factor X a showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor X a conformational change. The purpose of this paper is to extend these observations to bovine factor X a and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X a also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor X a. C6PS induced a 70-fold increase in bovine factor X a's autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X a. In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X a induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca 2+-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor X a elicits different structural and functional responses.