Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN′ by NMR
Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN′ by NMR
复制标题
DOI：
10.1110/ps.0235203
复制
发表时间：
2003-04-01
期刊：
影响因子：
8
通讯作者：
Bachovchin, WW
中科院分区：
文献类型：
--
作者：
Day, RM;Thalhauser, CJ;Bachovchin, WW
我们用N-15、H-1和C-13 NMR测定了枯草杆菌蛋白酶BPN ′及其PMSF和肽硼酸复合物中六个组氨酸在水溶液中的化学行为，在5 - 11范围内，它们是pH值的函数，并对静息酶中所有组氨酸侧链的每一个N-15、H-1、C-δ 1和C-δ 2共振进行了归属。6个组氨酸残基中的4个（17、39、67和226）带中性电荷，不滴定。一个组氨酸（238）位于蛋白质表面，在25 ℃时具有pK（a）= 7.30 +/- 0.03，具有快速的质子交换，但限制了迁移率。双位点交换计算机线形模拟显示，突变体N155 A的活性位点组氨酸（64）的pK（a）值为7.9 ± 0.3，质子交换行为缓慢。His 64在静息酶中含有极高的C-ε 1-H质子化学位移，为百万分之9.30（ppm），这是由于保守的C-ε 1-（HO）-O-... = C H-键从活性位点咪唑到骨架羰基，这在代表所有四个超家族的所有已知丝氨酸蛋白酶中发现。只有His 226和His 64在高pH下以罕见的N-δ 1-H互变异构体存在，其C-13（δ 1）化学位移比N-ε 2-H互变异构体高约9 ppm。与静息酶不同，PMSF复合物中的His 64在其低pH形式下具有高度移动的性，如N-15-H-1 NOE效应所示，并且具有与pK（a）值7.47 +/- 0.02相关的快速质子交换动力学。
We have determined by N-15, H-1, and C-13 NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every N-15, H-1, C-delta1, and C-delta2 resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25degreesC, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/-0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C-epsilon1-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C-epsilon1-(HO)-O-... = C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N-delta1-H tautomer, exhibiting C-13(delta1) chemical shifts -9 ppm higher than those for N-epsilon2-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by N-15-H-1 NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.