ROLE OF HISTIDINE-64 IN THE CATALYTIC MECHANISM OF HUMAN CARBONIC ANHYDRASE-II STUDIED WITH A SITE-SPECIFIC MUTANT
ROLE OF HISTIDINE-64 IN THE CATALYTIC MECHANISM OF HUMAN CARBONIC ANHYDRASE-II STUDIED WITH A SITE-SPECIFIC MUTANT
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DOI：
10.1021/bi00445a054
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发表时间：
1989-09-19
期刊：
影响因子：
2.9
通讯作者：
LINDSKOG, S
中科院分区：
文献类型：
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作者：
TU, CK;SILVERMAN, DN;LINDSKOG, S
为了验证这一假设，即组氨酸64在人类碳酸酐酶II的活性位点的功能作为一个质子转移基团的催化CO2水合作用，我们研究了一个位点特异性突变体具有组氨酸64取代丙氨酸，不能转移质子。CO2水合的稳态动力学已经被测量，以及在化学平衡下CO2和水之间的18 O交换。结果表明，在pH > 7.0时，化学平衡时CO2和HCO 3-之间的交换速率基本上不受氨基酸取代的影响，而在pH < 7.0时，突变体中的交换速率略有降低（在pH 6.0时降低2倍）。然而，在不存在缓冲液的情况下，与未修饰的酶相比，突变体从含水底物氧的活性位点释放的速率小多达20倍。此外，在未修饰的酶中，水的释放被微摩尔浓度的Cu 2+离子抑制，但在丙氨酸64变体中没有观察到这种抑制。这些结果表明，该突变特别影响了活性位点和反应介质之间的质子转移速率。突变体中的这种动力学缺陷可以通过增加某些缓冲液（如咪唑和1-甲基咪唑）的浓度来克服，但不能通过其他缓冲液（如MOPS或HEPES）来克服。类似地，在MOPS或TAPS缓冲液存在下，丙氨酸64变体催化的稳态CO2水合的最大速率非常低，但在咪唑衍生物存在下显著更高。从这些结果中得出的主要结论是，组氨酸64在天然碳酸酐酶II的功能作为质子穿梭组，提供了一个有效的途径，质子之间的转移活性位点和缓冲液分子是解决方案。涉及金属位点和缓冲液之间的直接质子转移的替代途径也可以显著地促进总催化速率，但是该途径的功效取决于缓冲化合物的化学性质。
To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH > 7.0 and slightly decreased in the mutant at pH < 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by other buffers, such as MOPS or HEPES. similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives. The main conclusion derived from these results is that histidine 64 in native carbonic anhydrase II functions as a proton-shuttle group, providing an efficient pathway for the transfer of protons between the active site and buffer molecules is solution. An alternative pathway involving direct proton transfer between the metal site and buffer can also contribute significantly to the overall catalytic rate, but the efficacy of this pathway depends on the chemical nature of the buffer compound.