Shin Jong-Shik, Yu Myeong-Hee
Department of Chemical Engineering, PO Box 43121, Texas Tech University, Lubbock, TX 79424, USA.
J Mol Biol. 2006 Jun 2;359(2):378-89. doi: 10.1016/j.jmb.2006.03.045. Epub 2006 Apr 3.
The native form of serine protease inhibitors (serpins) is kinetically trapped in a metastable state, which is thought to play a central role in the inhibitory mechanism. The initial binding complex between a serpin and a target protease undergoes a conformational change that forces the protease to translocate toward the opposite pole. Although structural determination of the final stable complex revealed a detailed mechanism of keeping the bound protease in an inactive conformation, it has remained unknown how the serpin exquisitely translocates a target protease with an acyl-linkage unhydrolyzed. We previously suggested that the acyl-linkage hydrolysis is strongly suppressed by active site perturbation during the protease translocation. Here, we address what induces the transient perturbation and how the serpin metastability contributes to the perturbation. Inhibitory activity of alpha1-antitrypsin (alpha1AT) toward elastase showed negative correlations with medium viscosity and Stokes radius of elastase moiety, indicating that viscous drag directly affects the protease translocation. Stopped-flow measurements revealed that the change in the inhibitory activity is primarily caused by the change in the translocation rate. The native stability of alpha1AT cavity mutants showed a negative correlation with the translocation rate but a positive correlation with the acyl-linkage hydrolysis rate, suggesting that the two kinetic steps are not independent but closely related. The degree of active site perturbation was probed by amino acid nucleophiles, supporting the view that the changes in the acyl-linkage hydrolysis rate are due to different perturbation states. These results suggest that the active site perturbation is caused by local imbalance between a pulling force driving protease translocation and a counteracting viscous drag force. The structural architecture of serpin metastability seems to be designed to ensure the active site perturbation by providing a sufficient pulling force, so the undesirable hydrolytic activity of protease is strongly suppressed during the translocation.
丝氨酸蛋白酶抑制剂（serpins）的天然形式在动力学上被困于亚稳态，这被认为在抑制机制中起核心作用。serpin与靶标蛋白酶之间的初始结合复合物会发生构象变化，迫使蛋白酶向相反的极移动。尽管最终稳定复合物的结构测定揭示了使结合的蛋白酶保持无活性构象的详细机制，但serpin如何精确地使靶标蛋白酶移位而酰基连接不被水解仍不清楚。我们之前提出，在蛋白酶移位过程中，活性位点的扰动会强烈抑制酰基连接的水解。在此，我们探讨是什么引发了这种瞬时扰动以及serpin的亚稳态如何导致这种扰动。α1-抗胰蛋白酶（α1AT）对弹性蛋白酶的抑制活性与弹性蛋白酶部分的介质粘度和斯托克斯半径呈负相关，表明粘性阻力直接影响蛋白酶的移位。停流测量表明，抑制活性的变化主要是由移位速率的变化引起的。α1AT腔突变体的天然稳定性与移位速率呈负相关，但与酰基连接水解速率呈正相关，这表明这两个动力学步骤并非相互独立，而是密切相关。氨基酸亲核试剂探测了活性位点的扰动程度，支持了酰基连接水解速率的变化是由于不同扰动状态的观点。这些结果表明，活性位点的扰动是由驱动蛋白酶移位的拉力与抵消粘性阻力之间的局部不平衡引起的。serpin亚稳态的结构架构似乎经过设计，通过提供足够的拉力来确保活性位点的扰动，因此在移位过程中蛋白酶不期望的水解活性会被强烈抑制。
