Structural role of Gly(193) in serine proteases: investigations of a G555E (GLY193 in chymotrypsin) mutant of blood coagulation factor XI.
Structural role of Gly(193) in serine proteases: investigations of a G555E (GLY193 in chymotrypsin) mutant of blood coagulation factor XI.
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Gly(193) 在丝氨酸蛋白酶中的结构作用：凝血因子 XI 的 G555E（胰凝乳蛋白酶中的 GLY193）突变体的研究。
DOI：
10.1074/jbc.m402971200
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Bajaj,SPaul
中科院分区：
文献类型：
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作者：
Schmidt,AmyE;Ogawa,Taketoshi;Gailani,David;Bajaj,SPaul
在丝氨酸蛋白酶中，Gly193 高度保守，几乎没有例外。据报道，一名患有凝血丝氨酸蛋白酶因子 XI (FXI) 遗传性缺陷的患者是 Gly555 → Glu 取代的纯合子。 FXI 中的 Gly555 对应于胰凝乳蛋白酶中的 Gly193，这是随后使用的编号系统。为了研究 FXIG193E 的异常情况，我们表达并纯化了重组 FXIaG193E，将其激活为 FXIaG193E，并将其活性与野生型激活的 FXI (FXIaWT) 进行比较。 FXIaG193E 以约 300 倍减少的 kcat 和类似的 Km 激活 FIX，并以约 10 倍减少的 Kman 和适度减少的 kcat 水解合成底物。抗凝血酶和淀粉样蛋白 β-前体蛋白 Kunitz 结构域抑制剂 (APPI) 与 FXIaG193E 的结合分别受损约 8,000 倍和约 100,000 倍。氟磷酸二异丙酯对 FXIaG193E 的抑制作用比 FXIaWT 慢约 30 倍，对氨基苯甲脒（S1 位点探针）的亲和力比 FXIaWT 弱 6 倍。 NH2-Ile16 与活性丝氨酸蛋白酶中的 Asp194 形成盐桥，其氨甲酰化速率比 FXIaG193E 快 4 倍。这些数据表明FXIaG193E的未占据活性位点未完全形成，并且Glu193的酰胺N可能不指向氧阴离子孔。包含饱和量的对氨基苯甲脒导致FXIaWT和FXIaG193E的氨甲酰化率相当，表明所占据的活性位点具有接近正常的构象。因此，小合成底物或抑制剂的结合提供了足够的能量，使 Glu193 的酰胺 N 正确指向氧阴离子孔。同源建模还表明，FXIaG193E 无法结合抗凝血酶/APPI 或激活 FIX，部分原因是由于与 Glu193 的空间冲突而导致 S2' 位点的可及性受损。这些论点将适用于用非甘氨酸残基取代 Gly193 的其他丝氨酸蛋白酶。
In serine proteases, Gly193is highly conserved with few exceptions. A patient with inherited deficiency of the coagulation serine protease factor XI (FXI) was reported to be homozygous for a Gly555→ Glu substitution. Gly555in FXI corresponds to Gly193in chymotrypsin, which is the numbering system used subsequently. To investigate the abnormality in FXIG193E, we expressed and purified recombinant FXIaG193E, activated it to FXIaG193E, and compared its activity to wild type-activated FXI (FXIaWT). FXIaG193Eactivated FIX with ∼300-fold reducedkcatand similarKm, and hydrolyzed synthetic substrate with ∼10-fold reducedKmand modestly reducedkcat. Binding of antithrombin and the amyloid β-precursor protein Kunitz domain inhibitor (APPI) to FXIaG193Ewas impaired ∼8,000- and ∼100,000-fold, respectively. FXIaG193Einhibition by diisopropyl fluoro-phosphate was ∼30-fold slower and affinity forp-aminobenzamidine (S1 site probe) was 6-fold weaker than for FXIaWT. The rate of carbamylation of NH2-Ile16, which forms a salt bridge with Asp194in active serine proteases, was 4-fold faster for FXIaG193E. These data indicate that the unoccupied active site of FXIaG193Eis incompletely formed, and the amide N of Glu193may not point toward the oxyanion hole. Inclusion of saturating amounts ofp-aminobenzamidine resulted in comparable rates of carbamylation for FXIaWTand FXIaG193E, suggesting that the occupied active site has near normal conformation. Thus, binding of small synthetic substrates or inhibitors provides sufficient energy to allow the amide N of Glu193to point correctly toward the oxyanion hole. Homology modeling also indicates that the inability of FXIaG193Eto bind antithrombin/APPI or activate FIX is caused, in part, by impaired accessibility of the S2′ site because of a steric clash with Glu193. Such arguments will apply to other serine proteases with substitutions of Gly193with a non-glycine residue.