Markley J L, Ibañez I B
Biochemistry. 1978 Oct 31;17(22):4627-40. doi: 10.1021/bi00615a008.
Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.
在2H₂O中，通过在低pH值下加热或暴露于6M盐酸胍使牛胰凝乳蛋白酶原A可逆展开，几乎导致所有产生低场¹H NMR峰的氮结合氢发生交换，而组氨酸环Cε1氢没有明显交换。这些预交换程序使得利用250-MHz相关¹H NMR光谱能够分辨出两个峰，这两个峰归属于胰凝乳蛋白酶原A的两个组氨酸残基。将Cε1氢峰指定给组氨酸-40和-57是基于将天然酶原的NMR滴定曲线与二异丙基磷酰基衍生物的滴定曲线进行比较。用预交换的胰凝乳蛋白酶Aα溶液也分辨出了两个组氨酸Cε1 H峰。通过将游离酶的NMR滴定曲线与其与牛胰蛋白酶抑制剂（Kunitz）的复合物的滴定曲线进行比较，确定了胰凝乳蛋白酶Aα的组氨酸峰。酶原和酶中组氨酸-57以及酶原中组氨酸-40的NMR滴定曲线呈现两个拐点；额外的拐点归因于与相邻羧基的相互作用：组氨酸-57的情况是天冬氨酸-102，酶原中组氨酸-40的情况是天冬氨酸-194。在31℃的2H₂O中的牛胰凝乳蛋白酶原A中，组氨酸-57的pK'为7.3，天冬氨酸-102的pK'为1.4，组氨酸-40 - 天冬氨酸-194体系在pH 4.6和2.3处呈现拐点。在相同条件下的牛胰凝乳蛋白酶Aα中，组氨酸-57 - 天冬氨酸-102体系的pK'值为6.1和2.8，组氨酸-40的pK'为7.2。结果表明，在酶原和酶中，组氨酸-57的pK'高于天冬氨酸-102的pK'。酶原和活化酶之间催化中心的结构和性质存在显著差异。除了pK'值的差异外，组氨酸-57的化学位移在酶原中高度异常（去屏蔽0.6 ppm），活化后变为正常。这些变化可能解释了活化后催化活性增加的部分原因。胰凝乳蛋白酶Aα - 胰蛋白酶抑制剂（Kunitz）复合物中组氨酸-57的Cε1 H的¹H NMR化学位移在pH 3至9之间保持恒定，其值与猪胰蛋白酶 - 胰蛋白酶抑制剂复合物中的组氨酸-57的值相似[Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487 - 509]，表明两种复合物中的相互作用机制相似。
