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 determines whether a protease can bind Na⁺. Serine proteases with a Pro at this position are unable to bind Na⁺, but those with a Tyr or Phe can bind Na⁺. Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na⁺ is also known to stimulate the amidolytic activity 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 Tyr²²⁵ was replaced with a Pro. We found that NA⁺ stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa ~8-24-fold with apparent dissociation constants [K(d)(app)] of 37 and 182 mM in the presence and absence of Ca²⁺, respectively. In contrast, NA⁺ minimally affected the cleavage rate 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 ~32-fold. Ca²⁺ improved the reactivity of the mutant with antithrombin ~5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor 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 reaction buffer. These results suggest that (1) the loop including residue 225 (225-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 forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.