Tucker H M, Gerard R D
Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas, USA.
Eur J Biochem. 1996 Apr 1;237(1):180-7. doi: 10.1111/j.1432-1033.1996.0180n.x.
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue-type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy-terminal side of the cleavage site of recombinant human PAI-1. Using site-directed mutagenesis, the P1'-P5' residues (P1' is the first residue on the carboxy-terminal side of the protease cleavage site) of the wild-type PAI-1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor-2, antithrombin, alpha 2-antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue-type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI-1. Analysis of substitutions at positions P4' and P5' both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue-type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI-1. Apparently, the determinants of the rapid inhibitory specificity of PAI-1 are localized to the P1'-P5' region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI-1 for plasminogen activators.
纤溶酶原激活物抑制剂-1（PAI-1）是丝氨酸蛋白酶抑制剂超家族的成员，是尿纤溶酶原激活物和组织型纤溶酶原激活物的快速作用抑制剂。我们评估了重组人PAI-1裂解位点羧基末端侧反应中心残基的功能意义。使用定点诱变，将野生型PAI-1反应中心序列的P1'-P5'残基（P1'是蛋白酶裂解位点羧基末端侧的第一个残基）替换为纤溶酶原激活物抑制剂-2、抗凝血酶、α2-抗纤溶酶和蛋白酶连接蛋白I的相应序列。测定了变体对丝氨酸蛋白酶尿纤溶酶原激活物、组织型纤溶酶原激活物、纤溶酶和凝血酶的抑制速率常数。结果表明反应中心长度和序列在PAI-1对纤溶酶原激活物的抑制中起关键作用。对P4'和P5'位置取代的分析证实并扩展了我们之前的工作，证明这些残基与组织型纤溶酶原激活物之间存在有利的静电相互作用。没有一个变体在其他丝氨酸蛋白酶的抑制速率常数上显示出显著增加，这表明仅这些残基不足以赋予PAI-1蛋白酶特异性。显然，PAI-1快速抑制特异性的决定因素定位于反应中心的P1'-P5'区域，这些残基协同作用以产生PAI-1对纤溶酶原激活物的精确特异性。
