Sodium binding site of factor Xa: Role of sodium in the prothrombinase complex

The nature of residue 225 on a consensus loop in serine proteases determine s whether a protease can bind Na+. Serine proteases with a Pro at this posi tion are unable to bind Na+, but those with a Tyr or Phe can bind Na+. Fact or Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na+ is also known to stimulate the amidolytic activit y of FXa toward cleavage of small synthetic substrates, but the role of Na in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) wa s replaced with a Pro. We found that Na+ stimulated the cleavage rate of ch romogenic substrates by FXa or GDFXa similar to 8-24-foldwith apparent diss ociation constants [K-d(app)] of 37 and 182 mM in the presence and absence of Ca2+, respectively. In contrast, Na+ minimally affected the cleavage rat e of these substrates by the mutant, and no K-d(app) for Na+ binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na+ and impaired similar to 32-fold. Ca2+ improved the reactivity of the mutant with antithrombin simi lar to 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further stu dies with the wild-type prothrombinase complex revealed that FXa binds to f actor Va with a similar K-d(app) of 1.1-1.8 nM in the presence of Na+, K+, Li+, Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na+ in the reacti on buffer. These results suggest that (1) the loop including residue 225 (2 25-loop) is a Na+ binding site in FXa, (2) the Na+- and Ca2+-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na+-bound and Na+-free f orms of factor Xa in the prothrombinase complex can efficiently activate pr othrombin.