Kaslik G, Westler W M, Gráf L, Markley J L
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Arch Biochem Biophys. 1999 Feb 15;362(2):254-64. doi: 10.1006/abbi.1998.1035.
Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).
结构和生化研究表明，丝氨酸蛋白酶抑制剂可诱导其靶标丝氨酸蛋白酶发生结构重排。先前对一种丝氨酸蛋白酶抑制剂α1-蛋白酶抑制剂与重组大鼠胰蛋白酶突变体（天冬氨酸189突变为丝氨酸的突变体，D189S，其比野生型大鼠胰蛋白酶对自身水解更稳定）之间复合物的核磁共振研究，提供了有关该复合物中催化残基状态的信息：复合物中天冬氨酸102与组氨酸57之间的氢键保持完整，组氨酸57的光谱特性更类似于酶原而非活化酶（G. Kaslik等人，1997年，《生物化学》36卷，5455 - 5464页）。在此，我们报告重组大鼠胰蛋白酶D189S的组氨酸57在三种状态下的质子化和交换行为：酶原、活性酶以及与人α1-蛋白酶抑制剂形成的复合物，并将这些行为与牛胰凝乳蛋白酶原和α-胰凝乳蛋白酶的组氨酸57的类似行为进行比较。在这些研究中，组氨酸57的pKa是根据天冬氨酸102 - 组氨酸57二元组中组氨酸Hδ1质子的1H NMR信号的pH依赖性确定的，而活性位点这一部分的可及性是通过该信号在选择性饱和后的出现速率来衡量的。大鼠胰蛋白酶原D189S（酶原，pKa = 7.8 ± 0.1；希尔系数 = 0.86 ± 0.05）的活化使组氨酸57的pKa降低了1.1个单位，并使质子化过程具有协同性（活性酶，pKa = 6.7 ± 0.1；希尔系数 = 1.37 ± 0.08）。α1-蛋白酶抑制剂与胰蛋白酶D189S的结合导致组氨酸57的pKa值升高至高于酶原的水平，并导致质子化过程出现负协同性（复合物，pKa = 8.1 ± 0.1；希尔系数 = 0.70 ± 0.08），这与酶原的情况相同。尽管组氨酸57在酶原、活性酶和α1-蛋白酶抑制剂复合物中的pKa存在这些差异，但在所有三种状态下，组氨酸57 Hδ1质子的溶剂交换寿命在实验误差范围内是相同的（寿命 = 2至12.5毫秒）。在低pH（5.2）和高pH（10.0）时，温度在5至20摄氏度之间，牛α-胰凝乳蛋白酶、重组大鼠胰蛋白酶D189S以及大鼠胰蛋白酶D189S与人α1-蛋白酶抑制剂复合物的光谱中，组氨酸57 Hδ1质子的1H NMR信号的线宽相对较窄；然而，在α-胰凝乳蛋白酶与人α1-蛋白酶抑制剂复合物的光谱中，该峰更宽，并且仅在较低温度（5摄氏度）下才能很好地分辨。
