Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases.
Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases.
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碳酸氢盐作为质子供体参与含 Zn(II) 和 Co(II) 的碳酸酐酶的催化。
DOI：
10.1021/ja010301o
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发表时间：
2001
影响因子：
15
通讯作者：
Silverman,DN
中科院分区：
文献类型：
--
作者：
Tu,C;Tripp,BC;Ferry,JG;Silverman,DN
分析了人碳酸酐酶II的Co（II）取代突变体对co2和水之间的18o交换的催化作用，以显示H218O从活性位点释放的速率。通过质谱测定，这一速率依赖于质子向金属结合的18o标记氢氧化物的转移，并且在碳酸酐酶II的位点特异性突变中观察到，其中一个突出的质子穿梭残基His64被丙氨酸取代，丙氨酸不支持质子运输。随着碳酸氢盐浓度的增加，H218O的释放速率以饱和方式增加，达到最大值4 × 105s-1，这与质子从碳酸氢盐转移到Co（II）结合的氢氧化物一致。同样的含Zn（II）碳酸酐酶突变体H218O的释放速度比含Co（II）的酶小10倍，但co2和hco3的相互转化速度与含Co（II）的酶基本相同。这些数据以及溶剂氢同位素效应表明，转移质子的碳酸氢盐与酶中的钴结合。来自嗜热古菌methanosarcina的含Zn（II）碳酸酐酶在催化过程中增加碳酸氢盐浓度引起18o交换的增强，这表明这种野生型酶具有非常相似的碳酸氢盐给予质子的机制。
Catalysis of18O exchange between CO2and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H218O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound18O-labeled hydroxide, and was observed in a site-specific mutant of carbonic anhydrase II in which a prominent proton shuttle residue His64 was replaced by alanine, which does not support proton transport. Upon increasing the concentration of bicarbonate, the rate of release of H218O increased in a saturable manner to a maximum of 4 × 105s-1, consistent with proton transfer from bicarbonate to the Co(II)-bound hydroxide. The same mutant of carbonic anhydrase containing Zn(II) had the rate of release of H218O smaller by 10-fold, but rate of interconversion of CO2and HCO3-about the same as the Co(II)-containing enzyme. These data as well as solvent hydrogen isotope effects suggest that the bicarbonate transferring the proton is bound to the cobalt in the enzyme. The enhancement of18O exchange caused by increasing bicarbonate concentration during catalysis by the Zn(II)-containing carbonic anhydrase from the archaeonMethanosarcina thermophilasuggests that a very similar mechanism for proton donation by bicarbonate occurs with this wild-type enzyme.