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

Journal Article (Journal Article)

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 Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), 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 approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 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 Ca(2+)-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.

Full Text

Duke Authors

Cited Authors

  • Rezaie, AR; He, X

Published Date

  • February 22, 2000

Published In

Volume / Issue

  • 39 / 7

Start / End Page

  • 1817 - 1825

PubMed ID

  • 10677232

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi992006a

Language

  • eng

Conference Location

  • United States