title: Molecular mechanism of dysfunctional factor VII associated with the homozygous missense mutation 331Gly to Ser.
abstract: We have identified a Japanese homozygous FVII deficiency associated with the mutation G331S (c184 [in chymotrypsin numbering]), and have determined the mechanisms responsible for the dysfunctional FVII variant by expressing the mutant recombinant FVII protein. In addition, the recombinant proteins FVIIG331D, G331W and G331F were expressed. The purified recombinant FVII proteins ran as a single chain form on SDS-PAGE having a molecular mass of approximately 50 Kda. The recombinant FVIIG331S expressed the level of the recombinant wild type FVII at 2.0%, and this mutant form was also similar to FVII in the patient's plasma. However, the amidolytic activity of FVIIa using peptidyl substrate S-2288 differed little between the wild type and the four mutant FVII molecules. We suggest that the functional defect found in these mutants is not directly associated with peptidyl substrate recognition or catalysis. The Km values of FX and FIX for the mutant proteins were approximately 7.6- to 15-fold and 13- to 19-fold higher than those for the wild-type protein, respectively. Molecular modelling indicated that the side chain of the S331 mutant is oriented close to the side chain of D338 (c189) at the bottom of the specificity pocket of FVIIa. We show that the replacement of G331 with a serine likely results in a steric hindrance of macromolecular substrate binding, leading to a loss of FVIIa enzymatic activity.
authors.affiliation: Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto Asahi, Japan. itosamu@gipac.shinshu-u.ac.jp
authors.forename: Osamu
authors.initials: O
authors.lastname: Takamiya
authors.forename: Shinya
authors.initials: S
authors.lastname: Kimura
mesh_terms: D003001:Cloning, Molecular; D005167:Factor VII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005168:Factor VII Deficiency / Q000235:genetics*; D006720:Homozygote; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D005167:Factor VII