Identification and characterization of the sodium-binding site of activated protein C

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal cataly tic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s ) of APC has not been identified. Based on a recent study with thrombin, th e 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla doma inless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) re quired Na+ (or other alkali cations) for its amidolytic activity with disso ciation constant (K-d(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, how ever, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated t he cleavage rate 5-6-fold with K-d(app) = 2.3 +/- 0.3 mm. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclud e that the 221-225 loop is a Na+-binding site in APC and that an allosteric link, between the Na+ and Ca2+ binding loops modulates the structure and f unction of this anticoagulant enzyme.