The effects of reactive site location on the inhibitory properties of the serpin α1-antichymotrypsin
The effects of reactive site location on the inhibitory properties of the serpin α1-antichymotrypsin
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DOI：
10.1074/jbc.m202374200
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发表时间：
2002-08-16
影响因子：
4.8
通讯作者：
Schechter, NM
中科院分区：
文献类型：
--
作者：
Plotnick, MI;Rubin, H;Schechter, NM
丝氨酸蛋白酶抑制剂反应位点环（RSL）的大尺寸表明，RSL在蛋白酶抑制中的作用比将反应位点（P1残基）呈递给蛋白酶的作用更复杂。本研究检查了将丝氨酸蛋白酶抑制剂α（1）-抗胰凝乳蛋白酶的反应位点（Leu-358）从RSL（Glu-342）的碱基靠近（P2）或远离（P1 '）一个残基的位置重新定位对抑制的影响。α（1）-抗胰凝乳蛋白酶变体通过P4-P2'区域内的突变产生;将序列ITLLSA改为ITLSSA以将反应位点重新定位到P2（Leu-357），并改为ITITTLS以将其重新定位到P1'（Leu-359）。分析糜蛋白酶样蛋白酶人糜蛋白酶和糜蛋白酶以及非靶蛋白酶人中性粒细胞弹性蛋白酶（HNE）的抑制。P2变体抑制糜蛋白酶和糜蛋白酶，但不抑制HNE。相对于P1，在P2的相互作用的特点是更大的复杂的稳定性，较低的抑制速率常数，并增加化学计量的抑制值。相反，P1'变体抑制HNE（抑制的化学计量= 4），但不抑制糜蛋白酶或糜蛋白酶。然而，HNE的抑制作用是通过与P2残基Ile-357相互作用实现的。P1'位点被所有蛋白酶识别为切割位点。在所有抑制反应中观察到对SDS-PAGE具有抗性的共价复合物，这与作为丝氨酸蛋白酶抑制剂-酰基蛋白酶复合物的蛋白酶的捕获一致。与Glu-342-反应位点距离由单个残基延长相关的抑制活性的完全丧失和与该距离由单个残基缩短相关的复合物的增强的稳定性与需要将RSL完全插入丝氨酸蛋白酶抑制剂的体内并将连接的蛋白酶易位到与遭遇相反的极的扭曲的蛋白酶抑制模型是相容的。
The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.