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J. Biol. Chem., Vol. 283, Issue 27, 18627-18635, July 4, 2008

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The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly^*

Raffaella Toso, Hua Zhu, and Rodney M. Camire¹

From the Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia and University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104

Zymogens of the chymotrypsin-like serine protease family are converted to the protease state following insertion of a newly formed, highly conserved N terminus. This transition is accompanied by active site formation and ordering of several surface loops in the catalytic domain. Here we show that disruption of this transition in factor X through mutagenesis (FXa^I16L and FXa^V17A) not only alters active site function, but also significantly impairs Na⁺ and factor Va binding. Active site binding was improved in the presence of high NaCl or with saturating amounts of factor Va membranes, suggesting that allosteric linkage exists between these sites. In line with this, irreversible stabilization of FXa^I16L with Glu-Gly-Arg-chloromethyl ketone fully rescued FVa binding. Furthermore, the K_m for prothrombin conversion with the factor Xa variants assembled into prothrombinase was unaltered, whereas the k_cat was modestly reduced (3- to 4-fold). These findings show that intramolecular activation of factor X following the zymogen to protease transition not only drives catalytic site activation but also contributes to the formation of the Na⁺ and factor Va binding sites. This structural plasticity of the catalytic domain plays a key role in the regulation of exosite expression and prothrombinase assembly.

Received for publication, March 19, 2008 , and in revised form, April 8, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants P01 HL-74124-01, Project 2 (to R. M. C.). This work was also supported by National Research Service Award T32 HL-07439-26 (to R. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Division of Hematology, 302E Abramson Research Center, The Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104. Tel.: 215-590-9968; Fax: 215-590-3660; E-mail: rcamire{at}mail.med.upenn.edu.

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