Structural and kinetic analysis of the chemical rescue of the proton transfer function of carbonic anhydrase II
Structural and kinetic analysis of the chemical rescue of the proton transfer function of carbonic anhydrase II
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DOI：
10.1021/bi002295z
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发表时间：
2001-02-13
期刊：
影响因子：
2.9
通讯作者：
McKenna, R
中科院分区：
文献类型：
--
作者：
Duda, D;Tu, CK;McKenna, R
人碳酸酐酶 II (HCA II) 中的组氨酸 64 在 CO2 水合的催化途径中发挥作用，作为在锌结合水和本体水之间转移质子的穿梭机。通过质谱法测量，CO2 和水之间 O-18 交换的催化依赖于这种质子转移，并且与野生型 HCA II 相比，H64A HCA II 的催化作用降低了 10 倍以上。 H64A HCA II 催化活性的丧失可以通过外源质子供体 4-甲基咪唑 (4-MI) 在饱和过程中恢复，最大活性为野生型 HCA II 的 40%。挽救的复合物的晶体结构在 1.6 埃分辨率下显示，4-MI 通过与 Trp 5 的 pi 堆积相互作用以及与水分子的氢键相互作用结合在 H64A HCA II 的活性位点空腔中。在此位置，4-MI距锌约12埃，接近观察到的野生型酶结构中His 64的“out”位置。 4-MI 似乎补偿了 His 64 的缺失并恢复了 H64A HCA II 突变体的催化活性。这一结果强烈表明，His 64 的外构象对于质子在锌结合溶剂分子和溶液之间的转移是有效的。
Histidine 64 in human carbonic anhydrase II (HCA II) functions in the catalytic pathway of CO2 hydration as a shuttle to transfer protons between the zinc-bound water and bulk water. Catalysis of the exchange of O-18 between CO2 and water, measured by mass spectrometry, is dependent on this proton transfer and was decreased more than 10-fold for H64A HCA II compared with wild-type HCA II. The loss of catalytic activity of H64A HCA II could be rescued by 4-methylimidazole (4-MI), an exogenous proton donor, in a saturable process with a maximum activity of 40% of wild-type HCA II. The crystal structure of the rescued complex at 1.6 Angstrom resolution shows 4-MI bound in the active-site cavity of H64A HCA II, through pi stacking interactions with Trp 5 and H-bonding interactions with water molecules. In this location, 4-MI is about 12 Angstrom from the zinc and approximates the observed "out" position of His 64 in the structure of the wild-type enzyme. 4-MI appears to compensate for the absence of His 64 and rescues the catalytic activity of the H64A HCA II mutant. This result strongly suggests that the out conformation of His 64 is effective in the transfer of protons between the zinc-bound solvent molecule and solution.