Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.
Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.
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在捕获目标蛋白酶期间，抗凝血酶的反应键环插入抑制剂的 Aβ-片层的免疫学证据。
DOI：
10.1021/bi00077a002
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Olson,ST
中科院分区：
文献类型：
--
作者：
Björk,I;Nordling,K;Olson,ST
1993年5月6日收到的修订版摘要：相同或高度相似的抗原决定簇，不存在于完整的抑制剂中，在抗凝血酶中诱导反应键断裂，在抗凝血酶和合成的反应环十四肽之间形成复合物，以及在低浓度的氯化胍下抗凝血酶部分变性。以前的研究表明，这三种修饰形式的抗凝血酶的共同结构特征是反应键的氨基末端侧的反应键环区域或相应的合成肽作为中间链插入抑制剂的主/3-片层A片层中。因此，三种修饰的抗凝血酶形式中的新表位最有可能由于这种插入而暴露。相同或高度相似的表位也暴露在抗凝血酶和凝血酶或Xa因子之间的复合物中，强烈表明反应性结合环的实质性片段也插入到这些复合物中的A片层中。相反，新的表位没有暴露在抗凝血酶肝素结合，这意味着肝素诱导的构象变化不涉及这样的环插入。这些结果为最近的假设提供了第一个实验验证，即丝氨酸蛋白酶抑制剂的反应性键环插入A/3-折叠中参与靶蛋白酶的结合。（1989）和Olson和Bjork（1992）]，是蛋白质的丝氨酸蛋白酶抑制剂超家族的成员（Carrell和Travis，1985）。除抗凝血酶外，该家族还包含大多数其他血浆丝氨酸蛋白酶抑制剂，例如α 1-蛋白酶抑制剂、α 1-抗胰凝乳蛋白酶、α 2-抗纤溶酶、Cl-抑制剂和纤溶酶原激活物抑制剂-1，以及几种非抑制性蛋白质，例如卵清蛋白和血管紧张素原（Huber & Carrell，1989）。抑制性丝氨酸蛋白酶抑制剂对靶蛋白酶的失活是由蛋白酶与抑制剂的特异性反应键相互作用引发的，并受到与抑制剂构象变化相关的后续捕获机制的影响。这种捕获导致形成动力学稳定的、可能共价连接的丝氨酸蛋白酶抑制剂-蛋白酶复合物，该复合物缓慢解离成无活性的、反应性键断裂的抑制剂和游离酶（Travis & Salvesen，1983; Carrell &博斯韦尔，1986; Huber & Carrell，1989; Bode & Huber，1992; Olson & Bjork，1992）。因此，丝氨酸蛋白酶抑制剂的作用机制与丝氨酸蛋白酶的低分子量蛋白质类似物的作用机制有很大不同（Laskowski & Kato，1980; Bode & Huber，1992）。捕获反应的性质一直是相当可观的猜测的主题，但仍然是未知的。已经报道了活性丝氨酸蛋白酶抑制剂的NoX射线结构，尽管几种非活性抑制剂的结构，其在反应键处或附近被切割（α 1-蛋白酶抑制剂，
Revised Manuscript Received May 6, 1993 abstract: Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced inantithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the aminoterminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main/3-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexesbetween antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A/3-sheet is involved in the binding of target proteinases.Antithrombin, the major plasma inhibitor of coagulation proteinases [for reviews, see Bj6rk et al.(1989) and Olson and Bjork (1992)], is a member of the serpin superfamily of proteins (Carrell & Travis, 1985). Besides antithrombin, this family also contains most other plasma serine proteinase inhibitors, eg, ai-proteinase inhibitor, ai-antichymotrypsin, a2-antiplasmin, Cl-inhibitor, and plasminogenactivator inhibitor-1, as well as several noninhibitory proteins, such as ovalbumin and angiotensinogen (Huber & Carrell, 1989). The inactivation of a target proteinase by an inhibitory serpin is initiated by the proteinase interactingwith a specific reactive bond of the inhibitor and is effected by a subsequent trapping mechanism associated with a conformational change of the inhibitor. This trapping leads to the formation of a kinetically stable, possibly covalently linked, serpin-proteinase complex that slowly dissociates to inactive, reactive-bond-cleaved inhibitor and free enzyme (Travis & Salvesen, 1983; Carrell & Boswell, 1986; Huber & Carrell, 1989; Bode & Huber, 1992; Olson & Bjork, 1992). The mechanism of action of serpins thus differs substantially from that of low-molecularweight protein inhibitorsof serine proteinases (Laskowski & Kato, 1980; Bode & Huber, 1992). The nature of the trapping reaction has been the subject of appreciable speculation but is still unknown. NoX-ray structure of an active serpin has been reported, although the structures of several inactive inhibitors, which either are cleaved at or near the reactive bond (ai-proteinase inhibitor,
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[2] 用对硝基苯基对-胍基苯甲酸酯HCl滴定胰蛋白酶、纤溶酶和凝血酶☆
DOI：
10.1016/0076-6879(70)19004-5
发表时间：
1970
期刊：
影响因子：
--
作者：
T. Chase;E. Shaw
通讯作者：
E. Shaw
影响因子：
64.8
作者：
CARRELL, RW;EVANS, DL;STEIN, PE
通讯作者：
STEIN, PE
DOI：
10.1016/s0021-9258(18)42309-5
发表时间：
1992-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
S. Olson;I. Björk;R. Sheffer;P. Craig;J. Shore;J. Choay
通讯作者：
S. Olson;I. Björk;R. Sheffer;P. Craig;J. Shore;J. Choay
影响因子：
3.9
作者：
MOUREY, L;SAMAMA, JP;MORAS, D
通讯作者：
MORAS, D
影响因子：
5.6
作者：
BAUMANN, U;HUBER, R;LAURELL, CB
通讯作者：
LAURELL, CB