Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1
Inhibitory specificity of the anti-inflammatory myxoma virus serpin, SERP-1
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DOI：
10.1074/jbc.273.33.20982
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发表时间：
1998-08-14
影响因子：
4.8
通讯作者：
McFadden, G
中科院分区：
文献类型：
--
作者：
Nash, P;Whitty, A;McFadden, G
SERP-1是粘液瘤病毒编码的丝氨酸蛋白酶抑制剂，由感染细胞分泌，是毒力所需，具有抗炎活性。我们报道了纯化的重组SERP-1与尿激酶型纤溶酶原激活剂（uPA）、组织型纤溶酶原激活剂（tPA）、纤溶酶、凝血酶和Xa因子形成SDS稳定的复合物，N端测序证实Arg（319）-Asn（320）为反应位点。这些残基突变为Ala-Ala消除了抑制活性，但对Thr（315）-Leu（316）的特异性切割没有影响。用弹性蛋白酶和组织蛋白酶G观察到，与uPA、tPA、纤溶酶反应的动力学分析，凝血酶、Xa和Cls显示二阶速率常数变化超过3 log，从凝血酶的k（inh）= 3 x 10（5）M（-1）s（-1）到Cls的600 M（-1）s（-1），而稳态抑制常数范围从凝血酶的K（I）= 10 pM到Cls的100 nM。抑制的化学计量在uPA的SI = 1.4 +/- 0.1至凝血酶的SI = 13 +/- 3之间变化。抑制动力学变化的分析表明，当丝氨酸蛋白酶抑制剂作用于以下离子时：浓度，与靶蛋白酶或K（I）相当（如对于体内SERP-1可能出现的那样），抑制特异性变得不那么受It支配，并且越来越依赖于分支反应机制内的分配和受抑制复合物的寿命。
SERP-1 is a myxoma virus-encoded serpin, secreted from infected cells, that is required for virulence and has anti-inflammatory activity. We report that purified recombinant SERP-1 forms SDS-stable complexes with urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, and factor Xa, N-terminal sequencing confirmed Arg(319)-Asn(320) as the site of reaction. Mutation of these residues to Ala-Ala abolished inhibitory activity but had no effect on the specific cleavage at Thr(315)-Leu(316) Seen with elastase and with cathepsin G, Kinetic analysis of the reactions with uPA, tPA, plasmin, thrombin, Xa, and Cls showed second-order rate constants to vary over 3 logs, from k(inh) = 3 x 10(5) M(-1) s(-1) with thrombin to similar to 600 M(-1) s(-1) with Cls, while steady-state inhibition constants ranged from K(I) = 10 pM with thrombin to similar to 100 nM with C1s. Stoichiometries of inhibition varied between SI = 1.4 +/- 0.1 for uPA to SI = 13 +/- 3 for thrombin, Analysis of the variations in inhibition kinetics shows that when serpins act at ion: concentrations, comparable with the target protease or with K(I) (as appears likely for SERP-1 in vivo), inhibitory specificity becomes less dominated by It,, and is increasingly dependent on partitioning within the branched reaction mechanism and on the lifetime of the inhibited complex.