Lysine-heparin interactions in antithrombin. Properties of K125M and K290M,K294M,K297M variants.
Lysine-heparin interactions in antithrombin. Properties of K125M and K290M,K294M,K297M variants.
复制标题
赖氨酸-肝素在抗凝血酶中的相互作用。
DOI：
10.1021/bi00251a026
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Gettins,PG
中科院分区：
文献类型：
--
作者：
Fan,B;Turko,IV;Gettins,PG
修订稿于 1994 年 9 月 7 日收到®摘要：抗凝血酶两个不同区域中的赖氨酸残基已被提议参与肝素结合和肝素介导的蛋白酶抑制加速。化学修饰研究表明赖氨酸 125 是一种重要的肝素结合残基 [Peterson, C. B.、Noyes, C. M.、Pecon, J. M.、Church, F. C. 和 Blackburn, M. N.(1987) J. Biol.化学。 262, 8061—8065] 而赖氨酸 290、294 和 297 已从模型构建研究中提出来构成肝素结合位点 [Villanueva, G. B.(1984) J. Biol. 262, 8061—8065]化学。 259、2531—2536]。为了评估这两个建议，我们制备了两种变体人类抗凝血酶，K125M和K290M、K294M、K297M，其中这些赖氨酸已通过定点诱变改变为蛋氨酸。 K290M、-K294M。 K297M变体在肝素亲和力以及凝血酶和因子Xa的抑制率方面具有与野生型重组抗凝血酶非常相似的特性。相反，K125M抗凝血酶对肝素五糖和全长肝素的亲和力均降低，对应于AAG 3.1和2。分别为0 kcal mol-1。然而，这种变体仍然能够抑制凝血酶和 Xa 因子。虽然凝血酶抑制率与野生型抗凝血酶相似，但 Xa 因子抑制率增强了 2 至 3 倍，表明赖氨酸 125 在肝素结合位点和反应中心区域之间的变构偶联中发挥作用。在肝素五糖或全长高亲和力肝素饱和时，K125M 和 K290M、K294M、K297M 变体的两种蛋白酶的抑制率与野生型抗凝血酶的抑制率相似。我们得出的结论是，赖氨酸 125 在肝素结合区的结构以及以高亲和力结合肝素方面发挥着重要作用，但对于最大程度地肝素诱导的蛋白酶抑制加速来说并不是必需的。我们没有发现明确的证据表明赖氨酸 290、294 和 297 有助于肝素结合位点，无论是作为主要位点还是参与结合较长链种类。抗凝血酶是丝氨酸蛋白酶抑制剂丝氨酸蛋白酶抑制剂超家族的成员，是参与凝血级联蛋白酶调节的蛋白酶抑制剂。抗凝血酶抑制的主要靶标是 Xa 因子和凝血酶。然而，为了有效抑制这些蛋白酶，肝素必须是
Revised Manuscript Received September 7, 1994® abstract: Lysine residues in two different regions of antithrombin have been proposed to be involved in heparin binding and heparin-mediated acceleration of proteinase inhibition. Lysine 125 has been implicated as an essential heparinbinding residue from chemical modification studies [Peterson, C. B., Noyes, C. M., Pecon, J. M., Church, F. C., & Blackburn, M. N.(1987) J. Biol. Chem. 262, 8061—8065] whereas lysines 290, 294, and 297 have been proposed from model building studies to constitute the heparin binding site [Villanueva, G. B.(1984) J. Biol. Chem. 259, 2531—2536]. To evaluate both of these proposals, we have prepared two variant human antithrombins, K125M and K290M, K294M, K297M, in which these lysines have been changed by site-directed mutagenesis to methionines. The K290M,-K294M. K297M variant had properties verysimilar to those of wild-type recombinant antithrombin in affinity for heparin, and in rates of inhibition of thrombin and factor Xa. In contrast, K125M antithrombin had reduced affinity for both heparin pentasaccharideand full-length heparin, corresponding to AAGs of 3.1 and2. 0 kcal mol-1, respectively. However, this variant was still able to inhibit both thrombin and factor Xa. Whereas therate of thrombin inhibition was similar to that of wild-typeantithrombin, the rate of factor Xa inhibition was enhanced between 2-and 3-fold, suggesting a role for lysine 125 in the allosteric coupling betweenthe heparin binding site and the reactive center region. At saturation with either heparin pentasaccharide or full-length high-affinity heparin, the rates of inhibition of both proteinases were similar to those of wild-typeantithrombin for both the K125M and K290M, K294M, K297M variants. We conclude that lysine 125 plays an important role in the structure of the heparin binding region and in binding heparin with high affinity, but is not needed for the maximum heparin-induced acceleration of proteinase inhibition. We found no definitive evidence that lysines 290, 294, and 297 contribute to a heparin binding site, either as the primary site or involved in binding longer chain species.Antithrombin is a member of the serpin superfamily of serine proteinase inhibitors and is an inhibitor of proteinases involved in regulation of proteinases of the blood coagulation cascade. The principal targets for inhibition by antithrombin are factor Xa and thrombin. However, for efficient rates of inhibition of these proteinases to occur, heparin must be