The Role of γ-COOH Glutamic Acids in the Enzymatic Functions of Factor Xa

In order to study the function of γ-carboxyglutamic acid residues (gla) in factor X(a) we compared the properties of factor X(a) and abnormal factor X(a) from anticoagulated cowl (PIVKA-X(a)), We studied Ca++ and phospholipid binding, spectral changes upon Ca++-binding and the activation by a specific fraction from+ Russells Viper venom (RW). In factor X but+ not in PIVKA-X a cooperative binding of about 5 Caions as well as a cooperative effect of Caions in spectral shift was observed.PIVKA-X can be converted into an active esterase by RVV. This reaction is Ca++ dependent. The Hill coefficient of this dependence is 2.8 for factor X and 1.0 for PIVKA-X. Factor Xa binds to micelles of an equimolar mixture of (dioleoyl) phosphatidyl-serine and dioleoyl phosphatidylcholine in the presence of Ca++ ions. Consistent with the inability of PIVKA-X to bind Ca++ ions cooperatively, PIVKA-X does not bind to phospholipid vesicles. Kinetic experiments on the activity of factor Xand PIVKA-X , in the absence of Ca+ions and phospholipids towards amides shows that both enzymes have identical Km values for each of the substrates but different kcat values. The results indicate a positive effect of gla residues on the rate of hydrolysis of amides. These studies suggest that the gla residues not only function to anchor factor X(a), on phospholipid via Ca++ ions, but that they also assist in obtaining conformational changes in the protein molecule as distant as to influence the active site. Indeed even the “anchor function” may depend upon such changes.