Maity H, Jarori G K
Tata Institute of Fundamental Research, Colaba, India.
Eur J Biochem. 1997 Dec 1;250(2):539-48. doi: 10.1111/j.1432-1033.1997.0539a.x.
The structure of adenine nucleotide bound at the active site of yeast hexokinase PII (PII) was studied in the complexes PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc using two-dimensional transferred NOE spectroscopy. Binding of the nucleotide ligand to the enzyme resulted in downfield shift of several ligand resonances. Changes in the chemical shift as a function of ligand concentration indicate that various resonances in the bound and free form of the ligand appear to be in fast exchange. The largest chemical shift change between the bound and the free states (delta vM = 88 +/- 9 Hz) at an NMR frequency of 500 MHz was observed for the H2 resonance of the adenine ring. A lower limit for the rate of ligand dissociation from the complex derived from these results is k(off) >> 550 s(-1). Interproton NOEs for various ligand protons in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes were measured at 500 MHz at 10 degrees C. The NOE buildup curves constructed from such measurements made at various mixing times (40, 80, 120, 160 and 200 ms) were analyzed using complete relaxation matrix calculations and various interproton distances were determined. These distances were used in restrained molecular dynamics calculations to derive the conformation of the nucleotide bound at the active site of the enzyme. The results of these calculations indicate that the nucleotide binds in an anti conformation. The glycosidic torsion angle chi (O4'-C1'-N9-C8) determined for the nucleotide in PII x ADPMg(II), PII x ADPMg(II) x Glc and PII x ADPMg(II) x NO3- x Glc complexes are 68 +/- 4 degrees, 52 +/- 4 degrees and 49 +/- 4 degrees respectively. In all these complexes, the ribose pucker is best represented by the unsymmetrical O4'-endo-C1'-exo twist ((o)T1). The observed decrease in the glycosidic torsion angle of the bound nucleotide is attributed to the glucose-induced conformational changes in the enzyme. The structure of the nucleotide derived here is at variance from the one proposed on the basis of X-ray crystallography and model building [Shoham, M. & Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14].
利用二维转移核Overhauser效应（NOE）光谱，对酵母己糖激酶PII（PII）活性位点结合的腺嘌呤核苷酸在PII×ADPMg（II）、PII×ADPMg（II）×葡萄糖和PII×ADPMg（II）×硝酸根×葡萄糖复合物中的结构进行了研究。核苷酸配体与酶的结合导致几个配体共振峰向低场移动。化学位移随配体浓度的变化表明，配体结合态和游离态的各种共振峰似乎处于快速交换状态。在500 MHz的核磁共振频率下，观察到腺嘌呤环H2共振峰在结合态和游离态之间的最大化学位移变化（δvM = 88±9 Hz）。根据这些结果推导得到的配体从复合物中解离的速率下限为k(off) >> 550 s(-1)。在10℃下，于500 MHz测量了PII×ADPMg（II）、PII×ADPMg（II）×葡萄糖和PII×ADPMg（II）×硝酸根×葡萄糖复合物中各种配体质子间的核Overhauser效应。由在不同混合时间（40、80、120、160和200 ms）进行的此类测量构建的NOE积累曲线，使用完整弛豫矩阵计算进行分析，并确定了各种质子间距离。这些距离用于受限分子动力学计算，以推导结合在酶活性位点的核苷酸的构象。这些计算结果表明，核苷酸以反式构象结合。在PII×ADPMg（II）、PII×ADPMg（II）×葡萄糖和PII×ADPMg（II）×硝酸根×葡萄糖复合物中测定的核苷酸糖苷扭转角χ（O4'-C1'-N9-C8）分别为68±4°、52±4°和49±4°。在所有这些复合物中，核糖的构象最好用不对称的O4'-内型-C1'-外型扭转（(o)T1）来表示。观察到的结合态核苷酸糖苷扭转角的减小归因于葡萄糖诱导的酶构象变化。这里推导得到的核苷酸结构与基于X射线晶体学和模型构建提出的结构不同[Shoham, M. & Steitz, T. A. (1980) J. Mol. Biol. 140, 1 - 14]。
