title: Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function.
abstract: To study the role of a putative high affinity Ca2+ binding site in the protease domain of factor X, we prepared a deletion mutant (E2FX) lacking the Gla and first epidermal growth factor-like domains. E2FX possesses a single high affinity Ca2+ binding site (Kd = 154 microM). Asp-70 or Glu-80 (chymotrypsin numbering system) was then converted to Lys. These mutants, E2FXD70K and E2FXE80K, allow examination of the Ca(2+)-dependent assembly of activation complexes involving substrate and enzyme forms of factor X that do not bind Ca2+. E2FXD70K and E2FXE80K share similar properties. They retain functional activity, but the D70K mutant no longer binds Ca2+, and neither mutant exhibits a Ca(2+)-dependent increase in peptide chromogenic substrate activity. Activation of E2FXD70K by the soluble tissue factor-factor VIIa complex was still Ca(2+)-dependent (Kd(app) = 133 +/- 38 microM), but the Kd(app) for Ca2+ was decreased relative to E2FX (Kd(app) = 205 +/- 53 microM). Prothrombin or prethrombin 1 activation by the E2FXaD70K in the presence of factor Va was also Ca(2+)-dependent (Kd(app) = 124 +/- 47 microM and 102 +/- 38 microM, respectively), and the Kd(app) was again lower than that of E2FXa (272 +/- 79 microM and 179 +/- 54 microM, respectively). In the absence of factor Va, activation of prethrombin 1 by E2FXaD70K was not influenced by Ca2+, but activation by E2FXa was enhanced (Kd(app) = 161 +/- 32 microM). The results suggest the presence of functionally important Ca2+ binding sites in components of the factor X and prothrombin activation complexes that do not involve factor X. Furthermore, the Ca2+ binding sites in factor X and trypsin appear to be structurally similar. The Asp or Glu-->Lys mutations within this site may create an internal salt bridge that conserves factor X (Xa) function even in the absence of Ca2+.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City.
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
authors.forename: C T
authors.initials: CT
authors.lastname: Esmon
mesh_terms: D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D001224:Aspartic Acid; D005170:Factor X; D015942:Factor VIIa; D008239:Lysine; D002118:Calcium