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

Publication ,  Journal Article
Rezaie, AR; He, X
Published in: Biochemistry
February 22, 2000

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.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 22, 2000

Volume

39

Issue

7

Start / End Page

1817 / 1825

Location

United States

Related Subject Headings

  • Transfection
  • Thromboplastin
  • Substrate Specificity
  • Sodium
  • Recombinant Proteins
  • Hydrolysis
  • Humans
  • Factor Xa Inhibitors
  • Factor Xa
  • Enzyme Precursors
 

Citation

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Rezaie, A. R., & He, X. (2000). Sodium binding site of factor Xa: role of sodium in the prothrombinase complex. Biochemistry, 39(7), 1817–1825. https://doi.org/10.1021/bi992006a
Rezaie, A. R., and X. He. “Sodium binding site of factor Xa: role of sodium in the prothrombinase complex.Biochemistry 39, no. 7 (February 22, 2000): 1817–25. https://doi.org/10.1021/bi992006a.
Rezaie AR, He X. Sodium binding site of factor Xa: role of sodium in the prothrombinase complex. Biochemistry. 2000 Feb 22;39(7):1817–25.
Rezaie, A. R., and X. He. “Sodium binding site of factor Xa: role of sodium in the prothrombinase complex.Biochemistry, vol. 39, no. 7, Feb. 2000, pp. 1817–25. Pubmed, doi:10.1021/bi992006a.
Rezaie AR, He X. Sodium binding site of factor Xa: role of sodium in the prothrombinase complex. Biochemistry. 2000 Feb 22;39(7):1817–1825.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

February 22, 2000

Volume

39

Issue

7

Start / End Page

1817 / 1825

Location

United States

Related Subject Headings

  • Transfection
  • Thromboplastin
  • Substrate Specificity
  • Sodium
  • Recombinant Proteins
  • Hydrolysis
  • Humans
  • Factor Xa Inhibitors
  • Factor Xa
  • Enzyme Precursors