Lawrence D A, Olson S T, Palaniappan S, Ginsburg D
Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0650.
J Biol Chem. 1994 Nov 4;269(44):27657-62.
One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
丝氨酸蛋白酶抑制剂（serpin）基因家族所有抑制性成员的一个共同特征是，其反应中心环的P14位置存在一个不带电荷的小残基。在本报告中，我们研究了丝氨酸蛋白酶抑制剂、纤溶酶原激活物抑制剂1型（PAI-1）中该位置突变的影响。将天然的P14苏氨酸-333残基替换为精氨酸（苏氨酸-333→精氨酸），导致对组织型纤溶酶原激活物和尿激酶型纤溶酶原激活物的抑制活性完全丧失。突变型抑制剂与野生型PAI-1（WTPAI-1）与脱水胰蛋白酶结合的比较表明，两种抑制剂与蛋白酶的初始相互作用是相同的。然而，虽然WTPAI-1与两种纤溶酶原激活物都形成了SDS稳定的复合物，但突变型PAI-1作为底物被有效切割。氨基末端序列分析表明，突变型PAI-1的切割发生在其反应中心的P1-P1'精氨酸-甲硫氨酸键处。天然和切割后的PAI的热变性研究表明，天然的苏氨酸-333→精氨酸突变体具有与活性WTPAI-1相同的热稳定性，并且两种蛋白质在被弹性蛋白酶切割后（在P4-P3键处切割）都变得明显更稳定。最后，分析了在P14处含有19种可能的氨基酸替代中的15种的重组PAI-1变体的功能。虽然残基大小似乎对抑制活性影响不大，但P14处正电荷或负电荷的存在会使PAI-1转化为底物。综上所述，这些结果表明，虽然反应中心环的插入对于蛋白酶结合不是必需的，但它是抑制剂功能所需的必要第二步。P14处带电荷残基的存在会阻碍这种插入，导致丝氨酸蛋白酶抑制剂转化为底物。
