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首页> 外文期刊>Biochemistry >THE ROLE OF THE INSERTION LOOP AROUND TRYPTOPHAN 148 IN THE ACTIVITY OF THROMBIN
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THE ROLE OF THE INSERTION LOOP AROUND TRYPTOPHAN 148 IN THE ACTIVITY OF THROMBIN

机译：TRYPTOPHAN 148周围的插入环在凝血酶活性中的作用

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摘要

Thrombin has trypsin-like specificity for Arg-Xaa and Lys-Xaa peptide bonds; however, it is much more specific than trypsin, cleaving far fewer peptide bonds in macromolecular substrates. To probe the nature of the specificity of thrombin, a mutant has been constructed in which the Trp(148) loop of thrombin has been replaced with the same loop of bovine trypsin. This mutant was expressed in Escherichia coli as prethrombin-21((148)) using a T7 expression system previously described for wild-type prethrombin-2 [DiBella et nl, (1995) J. Biol. Chem. 270, 163-169]. After refolding and purification, prethrombin-2((148)) was activated to thrombin((148)), with Echis carinatus snake venom, The k(cat)/K-m for the release of fibrinopeptide A from fibrinogen was 4.5 +/- 0.5 mu M(-1) s(-1) for thrombin((148)), which was similar to 20% of that of recombinant thrombin (25 +/- 2.0 mu M(-1) s(-1)). Thrombin ((148)), was inhibited less well by hirudin with a K-i of 500 pM compared to a value of 12 pM determined for recombinant thrombin. The mutant thrombin was also compared to trypsin and wild-type recombinant thrombin for the ability to cleave small peptide substrates. The Michaelis constants (K-m) were found to be between 5- and 10-fold higher for thrombin((148)), relative to wild-type recombinant thrombin, although the catalytic constants (k(cat)) for thrombin((148)) and recombinant thrombin remained relatively unchanged for all three substrates. Thrombin((148)) had a specificity constant (k(cat)/K-m) 2-fold higher for the hydrolysis of H-D-phenyalanyl-L-pipecolyl-L-arginine-p-nitroaniline (a thrombin substrate) than that of trypsin. For N-benzoyl-L-isoleucyL- L-glutamylglycyl-L-arginine-p-nitroaniline (a trypsin substrate) and N-carbobenzoxyglycylprolyl-L-arginine-p-nitroaniline (a substrate for both enzymes), the specificity constants for trypsin were 1000- and 16-fold higher, respectively, Although replacement of the Trp(148) loop does not yield an enzyme with more trypsinlike specificity, the Trp(148) loop is important in the substrate binding and specificity of thrombin (on the basis of K-m and K-i).
机译：凝血酶对Arg-Xaa和Lys-Xaa肽键具有胰蛋白酶样的特异性。然而，它比胰蛋白酶更具特异性，在大分子底物中裂解的肽键少得多。为了探测凝血酶特异性的性质，构建了一个突变体，其中的凝血酶的Trp（148）环已被相同的牛胰蛋白酶环取代。使用先前针对野生型凝血酶原-2描述的T7表达系统，该突变体在大肠杆菌中表达为凝血酶原21（（148））[DiBella等人，（1995）J.Biol.Chem.105：1897]。化学270，163-169]。重新折叠和纯化后，用纤毛虫蛇毒将凝血酶原2（（148））活化为凝血酶（（148）），从纤维蛋白原中释放纤维蛋白肽A的k（cat）/ Km为4.5 +/- 0.5凝血酶（（148））的mu M（-1）s（-1），类似于重组凝血酶（25 +/- 2.0μM（-1）s（-1））的20％。与重组凝血酶测定的12 pM值相比，水rud素的K-i为500 pM时对凝血酶（（148））的抑制作用较小。还将突变型凝血酶与胰蛋白酶和野生型重组凝血酶的裂解小肽底物的能力进行了比较。相对于野生型重组凝血酶，凝血酶（（148））的Michaelis常数（Km）高5至10倍，尽管凝血酶（（148））的催化常数（k（cat）） ），重组凝血酶对所有三种底物均保持相对不变。凝血酶（（148））的HD-苯丙氨酰-L-哌可基-L-精氨酸-对硝基苯胺（凝血酶底物）的水解特异性比常数（k（cat）/ Km）高2倍。对于N-苯甲酰基-L-异亮氨酸L-L-谷氨酰甘氨酰-L-精氨酸-对硝基苯胺（胰蛋白酶底物）和N-碳苯甲氧基甘氨酰脯氨酰基-L-精氨酸-对硝基苯胺（两种酶的底物），胰蛋白酶的特异性常数分别增加了1000倍和16倍，尽管替换Trp（148）环不会产生具有更多胰蛋白酶样特异性的酶，但是Trp（148）环在底物结合和凝血酶特异性方面很重要（基于Km和Ki）。

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