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

我们发现了一种新的功能障碍性凝血因子VII缺陷症，该缺陷症与凝血因子VII催化结构域中靠近底物结合位点的331 Gly到Ser的同源错义突变有关。为了研究G3331 S错义突变对FVII生物合成的影响，我们获得了重组FVII（野生型）和突变型FVII纯化的野生型FVII、突变型FVII 331 S、FVII 331 D、FVII 331 W和FVIIG 331 F分别含有142%、2.8%、5.0%、野生型VIIa和突变型VIIa对FVIIc的水解活性差异不大（FVIG331S，FVIG331D，FVIG 331 W和FVIG 331 F）具有相同的S-最大速度。2288水解。使用FXa的发色底物或FIXa的发色底物，在存在下研究了VIIa的大分子底物亲和力和催化作用。人TF和Ca. FX对野生型VIIa的Km和Vmax分别为33 nM和0.0143/min。日 ...更多信息 VIIa型、FVIG 331 S、FVIG 331 D、FVIG 331 W和FVIG 331 F的速率分别为250 nM和0.005/min、143 nM和0.0033/min、500 nM和0.0017/min和500 nM和0.0018/min。FX在突变型VIIa中的Km约为野生型VIIa的7.6- 15倍，野生型VIIa对FIX的Km和Vmax分别为26 nM和0.0083/min。VIIa型、FVIG 331 S、FVIG 331 D、FVIG 331 W和FVIG 331 F的这些分别为340 nM和0.0065/min、300 nM和0.0066/min、500 nM和0.0017/min以及500 nM和0.0017/min。在分子图形中，酶催化结构域中的Gly 331位于Cys 310-Cys 329之间，Asp 338是酶底物结合口袋底部的关键残基。它似乎主要在保持Gly 331侧链和Asp 338侧链之间的有利距离方面起作用。这表明FVIG 331 S、FVIG 331 D、FVIG 331 W和FVIG 331 F的功能缺陷与肽基FVII底物的抑制或催化不直接相关。它们可能引起底物结合的空间位阻，导致酶活性的丧失。少

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