Blouse Grant E, Perron Michel J, Thompson Jannah H, Day Duane E, Link Chad A, Shore Joseph D
Henry Ford Health Sciences Center, Division of Biochemical Research, One Ford Place 5-D, Detroit, MI 48202, USA.
Biochemistry. 2002 Oct 8;41(40):11997-2009. doi: 10.1021/bi025967p.
The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.
丝氨酸蛋白酶抑制剂（serpins）的抑制机制需要结构发生变化，以将靶蛋白酶捕获为稳定的酰基 - 酶复合物。尽管通常认为反应中心环插入及相关的构象变化是协同进行的，但这尚未得到直接证明。通过用7 - 氮杂色氨酸取代色氨酸并分析瞬态反应动力学，我们将抑制性丝氨酸蛋白酶抑制剂 - 蛋白酶复合物的形成描述为丝氨酸蛋白酶抑制剂结构的单一协同转变。用具有独特光谱的类似物7 - 氮杂色氨酸取代纤溶酶原激活物抑制剂1型（PAI - 1）的四个色氨酸，使得能够观察到丝氨酸蛋白酶抑制剂的构象变化，而非蛋白酶的构象变化。与组织型纤溶酶原激活剂（t - PA）或尿激酶（u - PA）形成共价酰基 - 酶复合物而非非共价米氏复合物，导致荧光迅速下降，这与反应中心环的插入和β - 折叠A的扩展同时发生。将由反应中心环的P14 - P7残基组成的八肽插入β - 折叠A中，通过7 - 氮杂色氨酸荧光测量，在丝氨酸蛋白酶抑制剂结构中产生了相同的构象变化，这表明近端环残基的引入诱导了丝氨酸蛋白酶抑制剂分子的结构重排。通过类似物取代对色氨酸吲哚环进行原子特异性修饰，对蛋白酶功能产生了特异性影响。PAI - 1对t - PA而非u - PA的抑制活性降低，表明环插入机制对一个或多个色氨酸残基的分子内相互作用敏感。
