Song S, Vander Velde D, Gunn C W, Himes R H
Department of Biochemistry, University of Kansas, Lawrence 66045.
Biochemistry. 1994 Jan 25;33(3):693-8. doi: 10.1021/bi00169a010.
ATP and ADP bind to N10-CHO-H4folate synthetase from Clostridium cylindrosporum at four identical sites. Although both ADP and ATP bind to the enzyme with essentially the same Ka values as the Mg2+.nucleotide complexes, only the Mg2+.nucleotides are kinetically active. Using transferred nuclear Overhauser effect (TRNOE) NMR spectroscopy, we have measured the time-dependent NOE buildup rates of selected protons in ADP and ATP bound to N10-CHO-H4folate synthetase after preirradiating protons H1', H2', H3', and H4'. The results were used to calculate interproton distances. In order to define the conformations of ADP and ATP bound to the enzyme, we used the TRNOE distance constraints in a distance geometry algorithm. The results of the distance geometry calculations suggest that, within experimental error, the conformations of both ADP and ATP (with or without Mg2+) have an average glycosidic torsion angle X (O4'-C1'-N9-C8) of 100 degrees +/- 20 degrees and a sugar pucker angle psi' (C5'-C4'-C3'-O3') of 85 degrees +/- 5 degrees. These values are consistent with a nucleotide structure generated by computer modeling after energy minimization, which has X = 90 degrees +/- 6 degrees and psi' = 81 degrees, indicating a high-anti and C3'-endo conformation.
ATP和ADP在四个相同的位点与柱状芽孢梭菌的N10-CHO-H4叶酸合成酶结合。虽然ADP和ATP与该酶结合时的解离常数（Ka）与Mg2⁺·核苷酸复合物基本相同，但只有Mg2⁺·核苷酸具有动力学活性。利用转移核Overhauser效应（TRNOE）核磁共振光谱，我们在对质子H1'、H2'、H3'和H4'进行预辐照后，测量了与N10-CHO-H4叶酸合成酶结合的ADP和ATP中选定质子的时间依赖性NOE积累速率。结果用于计算质子间距离。为了确定与该酶结合的ADP和ATP的构象，我们在距离几何算法中使用了TRNOE距离约束。距离几何计算结果表明，在实验误差范围内，ADP和ATP（有或没有Mg2⁺）的构象平均糖苷扭转角X（O4'-C1'-N9-C8）为100°±20°，糖环皱折角ψ'（C5'-C4'-C3'-O3'）为85°±5°。这些值与能量最小化后通过计算机建模生成的核苷酸结构一致，该结构的X = 90°±6°，ψ' = 81°，表明是高反式和C3'-内型构象。
