The structure of complex of sulfonamide antiglaucomatous agent with carbonic anhydrase as studied by NMR spectroscopy

为了获得抗癫痫药乙酰唑胺与碳酸酐酶Ⅱ、^1H、^ C和^N形成的复合物的结构信息<13><15>，进行了核磁共振研究。对于同位素标记的碳酸酐酶II的制备，将编码人碳酸酐酶II的cDNA转导到组氨酸和甘氨酸营养缺陷型的宿主细胞（大肠杆菌BL 21菌株）中。cDNA由另一个实验室提供。这些细胞是在我们自己的实验室里分离出来的。该酶从生长在含有14 <13>C和14 <15>N标记的组氨酸和甘氨酸的培养基中的细胞中分离，并通过亲和色谱和离子交换色谱进行纯化。根据报道的方法合成[乙酰胺-2-基]-<13>N，<15>N-二甲基-乙酰唑胺。进行以下NMR测量。它们是CO-过滤的1 <15>N-HSQC、3D-HNCA、2D-HCalphaC β H、（H）C β（CalphaC δ）H。通过这些程序，我们从His 64残基中获得NMR信号。然后，我们用~ 1C-编辑的NOESY方法测量了人碳酸酐酶II与乙酰唑胺复合物的NMR<13>，我们没有检测到乙酰唑胺的乙酰胺部分与酶的His 64残基之间的NOE（核Overhauser效应）相互作用。但我们确实检测到了该部分和一种疑似苯丙氨酸的残留物之间的反应。目前的结果不符合我们的假设，乙酰胺部分可能与酶的His 64残基的相互作用，虽然在得出最终结论之前需要进一步的研究。

In order to obtain information on the structure of the complex of acetazolamide, a potent antiglaucomatous agent, with carbonic anhydrase II,^1H,^<13>C and ^<15>N-nuclear magnetic resonance study was conducted. As for the preparation of isotope-labeled carbonic anhydrase II,cDNA encoding human carbonic anhydrase II was transduced into the host cells (BL21 strain of E.coli) which were auxotrophic for histidine and glycine. The cDNA was supplied from another laboratory. The cells were isolated in our own laboratory. The enzyme was separated from cells grown in a medium containing ^<13>C and ^<15>N-labeled histidine and glycine, and purified by both affinity and ion-exchange chromatography. [acetamide-2-^<13>C, ^<15>NJ-acetazolamide was synthesized according to a reported procedure. The following NMR measurements were carried out. They are CO-filtered ^<15>N-HSQC,3D-HNCA,2D-HCalphaCbetaH, (H) Cbeta (CalphaCdelta) H.Through these procedures, we distinguishd NMR signals from His64 residue. Then, we measured NMR of the complex of human carbonic anhydrase II with acetazolamide by means of ^<13>C-edited NOESY.We did not detect NOE (nuclear Overhauser effect) interaction between acetamide moiety of acetazolamide and His64 residue of the enzyme. but we did detect a reaction between the moiety and a residue which is suspected to be phenylalanine. The present results do not co-incide with our hypothesis that acetamide moiety may interact with His64 residue of the enzyme, although further study is required before coming to a final conclusion.