Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.
Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.
复制标题
丝氨酸蛋白酶中的酶原激活。
作者：
J. Markley;I. Ibáñez
通过在低 pH 下加热或暴露于 6 M 氯化胍，牛胰凝乳蛋白酶原 A 在 2H2O 中可逆解折叠，导致几乎所有氮结合氢发生交换，从而产生低场 1H NMR 峰，而组氨酰环 Cepsilon1 氢没有明显交换。这些预交换程序能够使用 250 MHz 相关 1 H NMR 光谱解析两个峰，这两个峰归因于胰凝乳蛋白酶原 A 的两个组氨酰残基。将 Cepsilon1 氢峰分配给组氨酸-40 和 -57 是基于天然酶原与二异丙基磷酰基衍生物的 NMR 滴定曲线的比较。两个组氨酰 Cepsilon1 H 峰也可以用预交换胰凝乳蛋白酶 Aalpha 溶液解析。通过比较游离酶与其与牛胰腺胰蛋白酶抑制剂（Kunitz）的复合物的NMR滴定曲线来确定胰凝乳蛋白酶Aα的组氨酰基峰。酶原和酶中的组氨酸57和酶原中的组氨酸40的NMR滴定曲线表现出两个拐点；额外的变化归因于与邻近羧基的相互作用：在组氨酸 57 的情况下为天冬氨酸 102，在酶原的组氨酸 40 的情况下为天冬氨酸 194。在 31°C 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 系统在 pH 4.6 和 2.3 时表现出变化。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系统的pK'值为6.1和2.8，组氨酸40的pK'值为7.2。结果表明，在酶原和酶中，组氨酸 57 的 pK' 均高于天冬氨酸 102 的 pK'。酶原和活化酶之间催化中心的结构和性质存在显着差异。除了 pK' 值的差异之外，酶原中高度异常的组氨酸 57 的化学位移（以 0.6 ppm 去屏蔽）在激活后变得正常化。这些变化可以部分解释活化后催化活性增加的原因。胰凝乳蛋白酶 Aα-胰蛋白酶抑制剂 (Kunitz) 复合物中组氨酸 57 的 Cepsilon1 H 的 1 H NMR 化学位移在 pH 3 和 9 之间恒定，其值与猪胰蛋白酶 - 胰蛋白酶抑制剂复合物中组氨酸 57 的值相似 [Markley, J.L. 和 Porubcan, M. A. (1976), J. Mol.生物。 102, 487--509]，表明这两种复合物的相互作用机制相似。
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.