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Disulfide locked variants of factor VIIa with a restricted -strand conformation have enhanced enzymatic activity

¹ Departments of Protein Engineering, ² Protein Chemistry, and ³ Physiology, Genentech Inc., South San Francisco, California 94080, USA⁴ Institute for Biology III, University of Freiburg, D-79104 Freiburg, Germany

(RECEIVED September 1, 2004; FINAL REVISION January 21, 2005; ACCEPTED January 31, 2005)

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of -strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF•FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V_max/K_m values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

Keywords: coagulation factor VIIa; tissue factor; serine protease; zymogen; hemostasis; disulfide; allostery

Abbreviations: TF, tissue factor • FVIIa, Factor VIIa • TF•FVIIa, tissue factor•Factor VIIa complex • FVII, Factor VII • FIX, Factor IX • FIXa, Factor IXa • FX, Factor X • FXa, Factor Xa • sTF, soluble tissue factor comprising the extracellular domain, residues 1–219 • pNA, para-nitroanalide

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041097505.

Reprint requests to: Robert A. Lazarus, Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA; e-mail: lazarus.bob{at}gene.com; fax: (650) 225-3734.

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