Mellet P, Boudier C, Mely Y, Bieth J G
Laboratoire d'Enzymologie, INSERM U 392, CNRS URA 491, Université Louis Pasteur de Strasbourg, Strasbourg F-67000, France.
J Biol Chem. 1998 Apr 10;273(15):9119-23. doi: 10.1074/jbc.273.15.9119.
Serpins are thought to inhibit proteinases by first forming a Michaelis-type complex that later converts into a stable inhibitory species. However, there is only circumstantial evidence for such a two-step reaction pathway. Here we directly observe the sequential appearance of two complexes by measuring the time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-alpha1-protease inhibitor. A moderately tight initial Michaelis-type complex EI1 (Ki = 0.38-0.52 microM) forms and dissociates rapidly (k1 = 1.5 x 10(6) M-1 s-1, k-1 = 0.58 s-1). EI1 then slowly converts into EI2 (k2 = 0.13 s-1), the fluorescence intensity of which is stable for at least 50 s. The two species differ by their donor-acceptor energy transfer efficiency (0. 41 and 0.26, respectively). EI2 might be the final product of the elastase + inhibitor association because its transfer efficiency is the same as that of a complex incubated for 30 min. The time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-eglin c, a canonical inhibitor, again allows the fast formation of a complex to be observed. However, this complex does not undergo any fluorescently detectable transformation.
丝氨酸蛋白酶抑制剂（Serpins）被认为首先通过形成米氏型复合物来抑制蛋白酶，该复合物随后会转化为稳定的抑制性物质。然而，对于这种两步反应途径仅有间接证据。在此，我们通过测量荧光素 - 弹性蛋白酶与罗丹明 - α1 - 蛋白酶抑制剂之间荧光共振能量转移的时间依赖性变化，直接观察到了两种复合物的相继出现。一种中等紧密的初始米氏型复合物EI1（Ki = 0.38 - 0.52微摩尔）形成并迅速解离（k1 = 1.5×10⁶ M⁻¹ s⁻¹，k⁻¹ = 0.58 s⁻¹）。然后EI1缓慢转化为EI2（k2 = 0.13 s⁻¹），其荧光强度至少在50秒内保持稳定。这两种物质的供体 - 受体能量转移效率不同（分别为0.41和0.26）。EI2可能是弹性蛋白酶 + 抑制剂结合的最终产物，因为其转移效率与孵育30分钟的复合物相同。荧光素 - 弹性蛋白酶与典型抑制剂罗丹明 - 埃格林c之间荧光共振能量转移的时间依赖性变化，再次使我们能够观察到复合物的快速形成。然而，这种复合物并未发生任何可通过荧光检测到的转化。
