Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase
Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase
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DOI：
10.1074/jbc.m609679200
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发表时间：
2007-03-30
影响因子：
4.8
通讯作者：
Kobayashi, Yuji
中科院分区：
文献类型：
--
作者：
Shimahara, Hideto;Yoshida, Takuya;Kobayashi, Yuji
组氨酸 64 (His(64)) 的咪唑 N-15 信号参与人碳酸酐酶 II (hCAII) 的催化功能，已被明确指定。这是通过掺入标记的组氨酸作为溶液 NMR 分析的探针来实现的，其中 N-15 在环 N-δ 1 和 N-ε 2 处，C-13 在环 C ε 1 处，C-13 和 N-15 在所有碳和氮处，或 N-15 在酰胺氮处，标记的甘氨酸在羰基碳处具有 C-13。利用环-N-15信号的pH依赖性以及实验曲线和模拟曲线之间的比较，我们确定His(64)的互变平衡常数(K-T)为1.0，这与其他组氨酸残基的互变平衡常数不同。这一独特的值将 His(64) 的咪唑氮原子表征为一般酸 (a) 和碱 (b)：其 ε 2-氮作为 (a) 将一个质子释放到本体中，而其 δ 1-氮作为来自水分子的另一个质子 (b) 提取与洞穴内锌结合水的水桥耦合。这加速了锌结合氢氧化物的生成，以与二氧化碳反应。从内部释放有效的碳酸氢根离子会分隔水桥路径，其中下一个水分子会移至锌离子旁边。新的水分子从 His(64) 的 delta 1-氮附近被供给。这些重建了水桥。基于这些特征，我们在此提出了hCAII的催化机制：His(64)的互变异构可以在中性pH下高效地介导质子和水分子的转移，不需要耗时或耗能的过程。
The imidazole N-15 signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with N-15 at ring-N-delta 1 and N-epsilon 2, C-13 at ring-C epsilon 1, C-13 and N-15 at all carbon and nitrogen, or N-15 at the amide nitrogen and the labeled glycine with C-13 at the carbonyl carbon. Using the pH dependence of ring-N-15 signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K-T) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon 2-nitrogen as (a) releases one proton into the bulk, whereas its delta 1-nitrogen as another proton from a water molecule within the (b) extracts water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta 1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.