ANALYSIS OF DYSFUNCTIOANAL FACTOR VII ASSOCIATED WITH HOMOZYGOUS MISSENSE MUTATION 331GLY TO SER

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We found the novel dysfunctional factor VII deficiency associated with homozygouse missense mutation 331Gly to Ser at close to substrate-binding site in catalytic domain of factor VII.To investigate the influence of the G3331S missense mutation on FVII biosynthesis, we obtained the recombinant FVII (wild type) and mutant FVII (FVIIG331S, FVIIG331D, FVIIG331W and FVIIG331F).The purified wild type FVII, mutant type FVII331S, FVII331D, FVII331W and FVIIG331F contained 142%,2.8%,5.0%,6.2% and 3.6% of FVIIc in normal pooled plasma, respectively.The amidolytic activity demonstrated little defference between wild-type VIIa and mutant-type VIIa (FVIIG331S, FVIIG331D, FVIIG331W and FVIIG331F) with identical maximal velocities of S-2288 hydrolysis.Macromolecular substrate affinity and catalysis of VIIa were studied using the chromgenic substrate for FXa or that for FIXa in presence of human TF and Ca. The observed Km and Vmax for FX for the wild-type VIIa was 33nM and 0.0143/min, resectively. Th … More ese of the mutant-type VIIa, FVIIG331S, FVIIG331D, FVIIG331W and FVIIG331F were 250nM and 0.005/min, 143nM and 0.0033/min, 500nM and 0.0017/min, and 500nM and 0.0018/min, respectively. The Km for FX the mutant-type VIIa was approximately 7.6-to 15-fold higher than those for wild-type VIIa.The observed Km and Vmax for FIX for the wild-type VIIa was 26nM and 0.0083/min, resectively. These of the mutant-type VIIa, FVIIG331S, FVIIG331D, FVIIG331W and FVIIG331F were 340nM and 0.0065/min, 300nM and 0.0066/min, 500nM and 0.0017/min, and 500nM abd 0.0017/min, and respectively. The Km for FIX the mutant-type VIIa was approximately 13-to 19-fold higher than those for wild-type VIIa.In the molecular graphics, the Gly331 in catalytic domain positioned between Cys310-Cys329 bound and Asp338,which was critical residue at the bottom of the substrate-binding pocket of the enzyme. It appeared to function primarily in maintaining a favorable distance between the side chain of Gly331 and Asp338 side chain. It demonstrate that the functional defect of FVIIG331S, FVIIG331D, FVIIG331W and FVIIG331F is not directly associated with peptidyl FVII substrate recongnition or catalysis. They would likely cause a steric hindrance of substrate binding, leading to the loss of enzymatic activity. Less