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Protein Science (2004), 13:431-442. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Zymogenic and enzymatic properties of the 70–80 loop mutants of factor X/Xa

Lin Chen1, Chandrashekhara Manithody1, Likui Yang and Alireza R. Rezaie

Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA

(RECEIVED September 2, 2003; FINAL REVISION October 15, 2003; ACCEPTED October 16, 2003)



Abstract

The Ca2+ binding 70–80 loop of factor X (fX) contains one basic (Arg71) and three acidic (Glu74, Glu76, and Glu77) residues whose contributions to the zymogenic and enzymatic properties of the protein have not been evaluated. We prepared four Ala substitution mutants of fX (R71A, E74A, E76A, and E77A) and characterized their activation kinetics by the factor VIIa and factor IXa in both the absence and presence of cofactors. Factor VIIa exhibited normal activity toward E74A and E76A and less than a twofold impaired activity toward R71A and E77A in both the absence and presence of tissue factor. Similarly, factor IXa in the absence of factor VIIIa exhibited normal activity toward both E74A and E76A; however, its activity toward R71A and E77A was impaired approximately two- to threefold. In the presence of factor VIIIa, factor IX activated all mutants with approximately two- to fivefold impaired catalytic efficiency. In contrast to changes in their zymogenic properties, all mutant enzymes exhibited normal affinities for factor Va, and catalyzed the conversion of prothrombin to thrombin with normal catalytic efficiencies. However, further studies revealed that the affinity of mutant enzymes for interaction with metal ions Na+ and Ca2+ was impaired. These results suggest that although charged residues of the 70–80 loop play an insignificant role in fX recognition by the factor VIIa-tissue factor complex, they are critical for the substrate recognition by factor IXa in the intrinsic Xase complex. The results further suggest that mutant residues do not play a specific role in the catalytic function of fXa in the prothrombinase complex.

Keywords: Factor X; factor Xa; factor VIIa; factor IXa; extrinsic Xase; intrinsic Xase

Abbreviations: fX, factor X • fXa, activated fX • R71A, E74A, E76A, and E77A, fX derivatives in which Arg71, Glu74, Glu76, and Glu77 in the chymotrypsinogen numbering system (Bode et al. 1989) have been replaced with Ala • fVIIa, active factor VII • TF, tissue factor • dcTF, TF in which cytoplasmic domain of the cofactor has been deleted • fIXa, active factor IX • fVIIIa, active factor VIII • fVa, active factor V • PEG, polyethylene glycol

Reprint requests to: Alireza R. Rezaie, Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA; e-mail: rezaiear{at}slu.edu; fax: (314) 977-7205.

1 These authors contributed equally to this study.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03406904.


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