GLU-192-]GLN SUBSTITUTION IN THROMBIN MIMICS THE CATALYTIC SWITCH INDUCED BY THROMBOMODULIN
GLU-192-]GLN SUBSTITUTION IN THROMBIN MIMICS THE CATALYTIC SWITCH INDUCED BY THROMBOMODULIN
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DOI：
10.1073/pnas.88.16.7371
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发表时间：
1991-08-01
影响因子：
11.1
通讯作者：
ESMON, CT
中科院分区：
文献类型：
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作者：
LEBONNIEC, BF;ESMON, CT
在丝氨酸蛋白酶中，残基 192（活性位点 Ser-195 之前的三个残基）在确定底物特异性方面发挥着重要作用。在胰蛋白酶 (EC 3.4.21.4) 和大多数具有相对广泛特异性的胰蛋白酶样酶中，该位置被 Gln 占据。在凝血酶 (EC 3.4.21.5) 中，一种具有有限特异性的酶，第 192 位被 Glu 占据。通过用 Gln 等排取代 Glu-192，研究了 Glu-192 在限制凝血酶特异性方面的潜在重要性。与胰蛋白酶不同，凝血酶对位置 P3 和 P'3 具有酸性残基的肽的裂解[其中 P3 和 P'3 分别指从氨基侧和羧基侧的 Arg (P1) 裂解位点去除的三个残基]效率较低。蛋白 C 是一种抗凝血酶原，在 P3 和 P'3 位有天冬氨酸残基。血栓调节蛋白是一种内皮细胞蛋白，与凝血酶复合可快速激活蛋白 C，从而改变凝血酶的特异性。与凝血酶相比，Glu-192 --> Gln 突变体凝血酶激活蛋白 C 的速度快 22 倍，从蛋白 C 激活位点切割 P7-P'5 肽的速度快 19 倍。蛋白 C 激活的增强主要是由于催化速率常数的增加，而不是米氏常数的提高，米氏常数是凝血酶-血栓调节蛋白复合物所共有的特性。 Glu-192 --> Gln 突变不影响纤维蛋白肽 A 的释放，仅使纤维蛋白肽 B 的释放速率增加 2.7 倍。这些结果表明，Glu-192 在限制凝血酶的特异性方面发挥着关键作用，并表明血栓调节蛋白可能部分通过改变凝血酶中残基 192 附近的酶-底物相互作用来发挥作用。
In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192 --> Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192 --> Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.