通过使用特定的氮-15标记酶来确认丝氨酸蛋白酶低场质子共振的归属。
Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.
作者信息
Bachovchin W W
出版信息
Proc Natl Acad Sci U S A. 1985 Dec;82(23):7948-51. doi: 10.1073/pnas.82.23.7948.
Abstract
Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field--i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the "charge-relay system" or catalytic triad of serine proteases [Robillard, G. & Shulman, R. G. (1972) J. Mol. Biol. 71, 507-511]. Since then, there have been a number of reports that have cast doubt on its correctness. In the present work we have tested this assignment using alpha-lytic protease (EC 3.4.21.12, Myxobacter alpha-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N delta 1 of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported [Robillard, G. & Shulman, R. G. (1974) J. Mol. Biol. 86, 519-540], which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N delta 1-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.
摘要
丝氨酸蛋白酶在(^{1}H_{2}O)溶液中的质子核磁共振谱通常在非常低的磁场下显示出单一共振峰——即相对于二甲基硅烷基戊烷磺酸盐为(14 - 18) ppm。该共振峰已被指定为丝氨酸蛋白酶“电荷中继系统”或催化三联体中天门冬氨酸 - 102和组氨酸 - 57（胰凝乳蛋白酶编号系统）之间氢键结合的质子[罗比拉德，G. 和舒尔曼，R. G.（1972年）《分子生物学杂志》71卷，507 - 511页]。从那时起，有许多报告对其正确性提出了质疑。在本研究中，我们使用α - 裂解蛋白酶（EC 3.4.21.12，粘细菌α - 裂解蛋白酶）对这一指定进行了测试，α - 裂解蛋白酶是一种与弹性蛋白酶同源的细菌丝氨酸蛋白酶，其单个组氨酸残基的(Nδ1)被氮 - 15特异性标记。这种标记酶的质子谱低场区域显示出一个具有所报道性质的单一共振峰[罗比拉德，G. 和舒尔曼，R. G.（1974年）《分子生物学杂志》86卷，519 - 540页]，此外，该共振峰对氮 - 15标记表现出自旋 - 自旋分裂。这种(^{15}Nδ1 - H)耦合的观察结果使得将该共振峰指定为电荷中继质子变得明确无疑。
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