Nishimura H, Takeya H, Miyata T, Suehiro K, Okamura T, Niho Y, Iwanaga S
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1993 Nov 15;268(32):24041-6.
Hemophilia B Fukuoka, a moderately severe bleeding disorder, is a naturally occurring mutant of factor IX. Plasma from our patient had 3% clotting activity even though 64% of factor IX antigen was present. The purified mutant protein was cleaved normally by factor Xla, factor VIIa-tissue factor complex, or RVV-X (factor X-activating enzyme from Russell's viper venom), yielding a two-chain factor IXa. Amino acid composition and sequence analyses of one of the lysyl endopeptidase peptides derived from factor IX Fukuoka revealed that Asn92 in the second epidermal growth factor (EGF)-like domain had been replaced by His. The active site of the factor IXa Fukuoka was normally competent for the incorporation of p-aminobenzamidine and for the hydrolysis of a synthetic substrate, N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester. Factor Xa formation by factor IXa Fukuoka was only 8% of the normal factor IXa, even in the presence of polylysine, and only 0.2% of the normal in the system containing phospholipids, Ca2+, and factor VIIIa, thereby indicating a functional defect in interaction of the mutant with factors VIIIa/X. Furthermore, catalytic efficiency (kcat/Km) of factor IXa Fukuoka toward factor X in the presence of Ca2+, phospholipids, and factor VIIIa was only 2.3% of the normal factor IXa. These results suggest that an Asn-to-His substitution at position 92 in the second EGF-like domain of factor IX Fukuoka would have an untoward effect on the specific conformational state of factor IX for binding with factors VIIIa/X.
血友病B福冈型是一种中度严重的出血性疾病，是因子IX的一种自然发生的突变体。我们患者的血浆中凝血活性仅为3%，尽管存在64%的因子IX抗原。纯化的突变蛋白能被因子Xla、因子VIIa-组织因子复合物或RVV-X（来自锯鳞蝰蛇毒的因子X激活酶）正常切割，产生双链因子IXa。对源自血友病B福冈型因子IX的一种赖氨酰内肽酶肽段进行氨基酸组成和序列分析表明，第二个表皮生长因子（EGF）样结构域中的Asn92被His取代。血友病B福冈型因子IXa的活性位点对于对氨基苯甲脒的掺入和合成底物Nα-苄氧羰基-L-精氨酸对硝基苄酯的水解通常是有活性的。即使在存在聚赖氨酸的情况下，血友病B福冈型因子IXa形成因子Xa的能力也仅为正常因子IXa的8%，而在含有磷脂、Ca2+和因子VIIIa的体系中仅为正常的0.2%，从而表明该突变体与因子VIIIa/X相互作用存在功能缺陷。此外，在存在Ca2+、磷脂和因子VIIIa的情况下，血友病B福冈型因子IXa对因子X的催化效率（kcat/Km）仅为正常因子IXa的2.3%。这些结果表明，血友病B福冈型因子IX第二个EGF样结构域第92位的Asn被His取代会对因子IX与因子VIIIa/X结合的特定构象状态产生不利影响。
