AMINO-ACID-RESIDUES THAT AFFECT INTERACTION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR WITH PLASMINOGEN-ACTIVATOR INHIBITOR-1
AMINO-ACID-RESIDUES THAT AFFECT INTERACTION OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR WITH PLASMINOGEN-ACTIVATOR INHIBITOR-1
复制标题
DOI：
10.1073/pnas.87.9.3530
复制
发表时间：
1990-05-01
影响因子：
11.1
通讯作者：
BASSELDUBY, RS
中科院分区：
文献类型：
--
作者：
MADISON, EL;GOLDSMITH, EJ;BASSELDUBY, RS
纤溶作用部分受组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物1(PAI-1，一种丝氨酸蛋白酶抑制物)相互作用的调节。我们早期的工作表明，t-PA丝氨酸蛋白酶结构域中一个氨基酸环(残基296-302)的缺失抑制了这两个蛋白质之间的相互作用，而不会改变t-PA对其底物纤溶酶原的反应活性。为了更准确地定义单个残基在这个环中的作用，我们使用了定点突变来用带负电荷的谷氨酸残基取代Lys-296、Arg-298和Arg-299。替换所有三个带正电的氨基酸会产生一种t-PA的变体，它与PAI-1结合效率低，并且高度抵抗丝氨酸的抑制。具有点突变的两个t-Pas(Arg-298.fwdarw.Glu和Arg-299.fwdarw。Glu)部分抵抗PAI-1的抑制，并以中等速率与丝氨酸结合。其他点突变(Lys-296.fwdarw.Glu，His-297.fwdarw.Glu和Pro-301。fwdarw。Gly)不会明显影响t-PA与PAI-1的相互作用。这些取代对t-PA的催化速率或酶与其底物纤溶酶原的亲和力都没有显著影响。在这些结果的基础上，我们提出了一个模型，即位于t=PA活性中心附近的表面环上的正电荷残基与PAI-1的活性中心C端互补的负电荷残基形成离子键。
Fibrinolysis is regulated in part by the interaction between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor of the serpin family). It is known from our earlier work that deletion of a loop of amino acids (residues 296-302) from the serine protease domain of t-PA suppresses the interaction between the two proteins without altering the reactivity of t-PA towards its substrate, plasminogen. To define more precisely the role of individual residues within this loop, we have used site-directed mutagenesis to replace Lys-296, Arg-298, and Arg-299 with negatively charged glutamic residues. Replacement of allthree positively charged amino acids generates a variant of t-PA that associates inefficiently with PAI-1 and is highly resistant to inhibition by the serpin. Two t-PAs with point mutations (Arg-298 .fwdarw. Glu and Arg-299 .fwdarw. Glu) are partially resistant to inhibition by PAI-1 and associate with the serpin at intermediate rates. Other point mutations (Lys-296 .fwdarw. Glu, His-297 .fwdarw. Glu, and Pro-301 .fwdarw. Gly) do not detectably affect the interaction of t-PA with PAI-1. None of these substitutions has a significant effect on the rate of catalysis by t-PA or on the affinity of the enzyme for its substrate, plasminogen. On the basis of these results, we propose a model in which positively charged residues located in a surface loop near the active site of t=PA form ionic bonds with complementary negatively charged residues C-terminal to the reactive center of PAI-1.