Chaillan-Huntington C E, Gettins P G, Huntington J A, Patston P A
Department of Oral Medicine and Diagnostic Sciences, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Biochemistry. 1997 Aug 5;36(31):9562-70. doi: 10.1021/bi970651g.
Two of the prototypic serpins are alpha1-proteinase inhibitor and ovalbumin. alpha1-Proteinase inhibitor is a rapid inhibitor of a number of proteinases and undergoes the characteristic serpin conformational change on cleavage within the reactive center loop, whereas ovalbumin is noninhibitory and does not undergo the conformational change. To investigate if residues from P12 to P2 in the reactive center loop of ovalbumin are intrinsically incapable of being in an inhibitory serpin, we have made chimeric alpha1-proteinase inhibitor variants containing residues P12-P7, P6-P2, or P12-P2 of ovalbumin and determined their inhibitory properties with trypsin and human neutrophil elastase. With the P12-P7 and P6-P2 variants, the steps before and after the fork of the branched suicide-substrate pathway were affected as reflected by changes in rates and stoichiometries of inhibition with both proteinases. The P12-P2 variant showed that those effects were nonadditive, with exclusive substrate behavior for elastase and only residual inhibitory activity against trypsin. The properties of the variants were consistent with them obeying the suicide-substrate mechanism characteristic of serpins. Enzyme activity was regenerated from complexes formed with the P6-P2 variant faster than with wild-type indicating that the rate of turnover of the complex was increased. Based on proteinase susceptibility in the reactive center loops of the P6-P2 and P12-P2 variants, and on an increase in heat stability of the cleaved P12-P2 variant, it was concluded that the variants had undergone complete loop insertion on cleavage. These results show that the reactive center loop residues P12-P2 of ovalbumin can be present in inhibitory serpins although decreasing the inhibitory properties. These data also demonstrate that the residues in the P6-P2 region of serpins are critical for rapid inhibition of proteinases and formation of stable serpin-proteinase complexes.
两种典型的丝氨酸蛋白酶抑制剂是α1-抗蛋白酶和卵清蛋白。α1-抗蛋白酶是多种蛋白酶的快速抑制剂，在反应中心环内被切割时会发生丝氨酸蛋白酶抑制剂特有的构象变化，而卵清蛋白无抑制作用且不会发生构象变化。为了研究卵清蛋白反应中心环中从P12到P2的残基是否本质上就无法形成具有抑制作用的丝氨酸蛋白酶抑制剂，我们构建了包含卵清蛋白P12 - P7、P6 - P2或P12 - P2残基的嵌合α1-抗蛋白酶抑制剂变体，并测定了它们对胰蛋白酶和人中性粒细胞弹性蛋白酶的抑制特性。对于P12 - P7和P6 - P2变体，分支自杀底物途径分叉前后的步骤受到影响，这在两种蛋白酶抑制的速率和化学计量变化中得到体现。P12 - P2变体表明这些影响并非叠加性的，对弹性蛋白酶表现出排他性底物行为，对胰蛋白酶仅具有残余抑制活性。这些变体的特性与它们遵循丝氨酸蛋白酶抑制剂特有的自杀底物机制一致。与P6 - P2变体形成的复合物比与野生型形成的复合物更快地恢复酶活性，这表明复合物的周转速率增加。基于P6 - P2和P12 - P2变体反应中心环对蛋白酶的敏感性，以及切割后的P12 - P2变体热稳定性的增加，得出结论：这些变体在切割后发生了完整的环插入。这些结果表明，卵清蛋白的反应中心环残基P12 - P2可以存在于具有抑制作用的丝氨酸蛋白酶抑制剂中，尽管会降低抑制特性。这些数据还表明，丝氨酸蛋白酶抑制剂P6 - P2区域的残基对于快速抑制蛋白酶和形成稳定的丝氨酸蛋白酶抑制剂 - 蛋白酶复合物至关重要。
