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

为了获得抗青光眼药物乙酰唑胺与碳酸酐酶II、^1H、^<13>C和^<15> n配合物的结构信息，进行了核磁共振研究。制备同位素标记的碳酸酐酶II，将编码人碳酸酐酶II的cDNA转导到对组氨酸和甘氨酸缺乏营养的大肠杆菌BL21株宿主细胞中。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。该酶从含有^<13>C和^<15> n标记的组氨酸和甘氨酸的培养基中培养的细胞中分离出来，并通过亲和层析和离子交换层析纯化。[acetamide-2-^<13>C, ^<15>NJ-acetazolamide]按报道的方法合成。进行了以下核磁共振测量。它们经过co过滤^<15>N-HSQC,3D-HNCA,2D-HCalphaCbetaH, (H) Cbeta (CalphaCdelta) H.通过这些程序，我们将核磁共振信号与His64残基区分开来。然后，我们用^<13> c编辑的noesi方法测量了人碳酸酐酶II与乙酰唑胺复合物的NMR。我们没有检测到乙酰唑胺的乙酰酰胺部分与酶的His64残基之间的NOE（核Overhauser效应）相互作用。但我们确实检测到了部分和疑似苯丙氨酸残留物之间的反应。目前的结果不符合我们的假设，即乙酰胺部分可能与酶的His64残基相互作用，尽管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.