Sette M, Paci M, Desideri A, Rotilio G
Department of Biology, University of Rome Tor Vergata, Italy.
Biochemistry. 1992 Dec 15;31(49):12410-5. doi: 10.1021/bi00164a016.
The binding of formate to bovine Cu,Zn superoxide dismutase has been studied by NMR spectroscopy. The distance between the copper ion and the proton covalently bound to formate has been evaluated from the broadening of the resonance of such proton. The effect on the copper-coordinated water molecule was evaluated from the bulk water relaxation effect by pulsed low-resolution NMR. The broadening of the resonance due to the formate carboxyl in the 13C NMR spectrum gave further indications about the carbon-copper distance thus providing information about the orientation of the formate ion. Changes of isotropically shifted resonances of the Cu,Co enzyme, where cobalt substitutes the native zinc, indicate that rearrangements of imidazoles of the liganding histidines occur upon binding. Transient NOE experiments gave indication of the proximity of the formate proton to resonance H of the NMR spectrum assigned to the imidazole proton of the copper-liganding His 118 of the active site. 2D NMR NOESY experiments made clear that no important rearrangement of the liganding histidines occurred in the presence of a saturating amount of formate. The absence of relevant changes of the intensity of NOE cross-peaks which are sensitive to interatomic distances in the active site revealed that only slight changes have occurred. Molecular graphics representation on the basis of all the information obtained allowed us to locate the formate in the proximity of the active site. The formate binding occurs via hydrogen bonds through the carboxylate ion and the NH groups of the side chains of Arg 141 which is external to the copper coordination sphere and faces the active site of the enzyme.
已通过核磁共振光谱研究了甲酸与牛铜锌超氧化物歧化酶的结合。通过与甲酸共价结合的质子共振峰的展宽来评估铜离子与该质子之间的距离。通过脉冲低分辨率核磁共振的体水弛豫效应来评估对铜配位水分子的影响。13C核磁共振谱中甲酸羧基引起的共振峰展宽进一步表明了碳 - 铜距离，从而提供了有关甲酸根离子取向的信息。在钴取代天然锌的铜钴酶中，各向同性位移共振的变化表明，结合时配体组氨酸的咪唑会发生重排。瞬态核Overhauser效应（NOE）实验表明，甲酸质子与核磁共振谱中归属于活性位点铜配体组氨酸118咪唑质子的共振峰H接近。二维核磁共振NOESY实验表明，在存在饱和量甲酸的情况下，配体组氨酸没有发生重要的重排。活性位点中对原子间距离敏感的NOE交叉峰强度没有相关变化，这表明只发生了轻微变化。基于所获得的所有信息进行的分子图形表示使我们能够将甲酸定位在活性位点附近。甲酸通过羧酸盐离子与位于铜配位球外部并面向酶活性位点的精氨酸141侧链的NH基团形成氢键而发生结合。
