The interaction of acetazolamide with carbonic anhydrase ----- An NMR study

用500 MHz~1H-核磁共振研究了碳酸氢酶II与目前用于治疗青光眼的乙酰唑胺的相互作用。主要研究结果如下：(1)以牛红细胞制备碳酸氢酶II为实验材料。用CPMG方法成功地检测了9个来自组氨酸残基C_2质子的信号。牛碳酸酐酶有11个组氨酸，但其中两个位于分子内。(2)我们尝试通过比较核磁共振测定的每个信号的脱氢率和FAB质谱仪测定的每个残基的脱氢率来确定这9个信号的一级结构。(3)与文献(1)相同的方法，我们研究了牛碳酸酐酶与乙酰唑胺的络合物，发现乙酰唑胺的乙酰胺部分除了与酶的其中一个组氨酸配位外，还与酶的必需锌离子配位。(4)除乙酰唑胺外，我们还研究了2-methylamino-1，3，4-thiadiazole-5-sulfonamide，的2-甲酰氨基-1，3，4-噻二唑-5-磺酰胺和磺胺。前两种化合物与乙酰唑胺形成了与碳酸酐酶类似的络合物，但磺胺没有。(5)为了确定与乙酰唑胺相互作用的残基的信号，我们尝试了羧甲基化His-，但这种羧甲基化也改变了光谱的其他重要部分。因此，我们尝试在X射线衍射法获得的坐标基础上进行分子力学研究。通过这一计算，我们认为His-可能是参与相互作用的残基的候选者。(6)在同工酶I的情况下，我们没有观察到我们在同工酶II的情况下检测到的这种相互作用。

Using 500 MHz ^1H-NMR we studied the interaction between carbonic anhydrase II and acetazolamide which is currently used for the therapy of glaucoma. The following results were obtained.(1) As the material, we employed carbonic anhydrase II prepared from bovine red blood cells. Using CPMG method, we succeeded in the detection of 9 signals derived from C_2 proton of histidine residues. Bovine carbonic anhydrase has 11 histidines but two of them are located inside the molecule.(2) We attempted to assign the 9 signals to the primary structure by means of comparison of the deuterization rate of each signal determined by NMR with the deuterization ratio of each residue determined by FAB mass spectrometry. However, we failed because we could not solve the problem of the noise due to FAB.(3) In the same way as described in (1), we studied the complex of bovine carbonic anhydrase with acetazolamide, and found that acetamide moiety of acetazolamide interacted with one of histidines of the enzyme in addition to the co-ordination of sulfamoyl moiety of acetazolamide to the essential zinc ion of the enzyme.(4) Other than acetazolamide, we also studied 2-methylamino-1,3,4-thiadiazole-5-sulfonamide, 2-formylamino-1,3,4-thiadiazole-5-sulfonamide and sulfanilamide. The former two compound formed a similar complex with carbonic anhydrase to acetazolamide, but sulfanilamide did not.(5) In order to assign the signal of the residue which interact with acetazolamide, we attempted to carboxymethylate His-64 but this carboxymethylation altered also other important part of the spectrum. Therefore we attempted molecular mechanic study based on the co-ordinate obtained by X-ray diffraction. By this calculation, we thought His-64 may be a candidate for the residue involved in the interaction.(6) In the case of isozyme I, we did not observe such an interaction as that we detected in the case of isozyme II.