Williams J S, Rosevear P R
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
J Biol Chem. 1991 Feb 5;266(4):2089-98.
Internuclear distances obtained from nuclear Overhauser effects were used in combination with a distance geometry algorithm to determine the conformation of Mg(alpha,beta-methylene)ATP bound to the Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) both in the absence and presence of cognate and noncognate amino acids. Mg(alpha,beta-methylene)ATP, a nonhydrolyzable analog of ATP, was used to prevent hydrolysis of the nucleotide in the presence of either cognate or noncognate amino acids. Kinetic analysis showed that Mg(alpha,beta-methylene)ATP was a linear competitive inhibitor with respect to ATP in the ATP-pyrophosphate exchange reaction with a Ki = 1.2 mM. The pattern of internuclear Overhauser effects on Mg(alpha,beta-methylene)ATP bound to delta MTS was qualitatively consistent only with an anti glycosidic torsional angle, suggesting that the adenosine portion of the nucleotide is uniquely oriented in the binary enzyme-nucleotide complex. Nearly identical patterns of nuclear Overhauser effects were also observed in ternary complexes containing either cognate L-methionine or noncognate L-homocysteine amino acids. Distance geometry calculations permitted the range and conformational space of the allowed adenine-ribose glycosidic torsional angles in each of the complexes to be better defined and compared. Average adenine-ribose glycosidic torsional angles for enzyme-bound Mg(alpha,beta-methylene)ATP of -106 +/- 9 degrees, -99 +/- 11 degrees, and -97 +/- 11 degrees were determined for the delta MTS.Mg(alpha,beta-methylene)ATP, delta MTS.Mg(alpha,beta-methylene)ATP.L-methionine, and delta MTS.Mg(alpha,beta-methylene)ATP.L-homocysteine complexes, respectively. Comparison of the three enzyme-bound conformations showed that a single nucleotide structure having an adenine-ribose glycosidic torsional angle of -98 degrees with a 3'-endo to O4'-exo ribose sugar pucker was, within error, consistent with the experimental internuclear distances obtained in all three complexes. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrates that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or the nature of the amino acid bound at the aminoacyladenylate site. Therefore, conformational changes known to occur in the methionyl-tRNA synthetase upon ligand binding appear not to alter the bound conformation of the nucleotide. Information on the conformation and arrangement of substrates bound at the aminoacyladenylate site of delta MTS is necessary for understanding the molecular mechanisms involved in amino acid activation and discrimination.
通过核Overhauser效应获得的核间距离与距离几何算法相结合，用于确定在不存在和存在同源及非同源氨基酸的情况下，与大肠杆菌截短的甲硫氨酰 - tRNA合成酶（δMTS）结合的Mg（α，β - 亚甲基）ATP的构象。Mg（α，β - 亚甲基）ATP是ATP的一种不可水解类似物，用于防止在存在同源或非同源氨基酸时核苷酸的水解。动力学分析表明，在ATP - 焦磷酸交换反应中，Mg（α，β - 亚甲基）ATP相对于ATP是线性竞争性抑制剂，Ki = 1.2 mM。对与δMTS结合的Mg（α，β - 亚甲基）ATP的核间Overhauser效应模式进行定性分析，仅与反式糖苷扭转角一致，这表明核苷酸的腺苷部分在二元酶 - 核苷酸复合物中具有独特的取向。在含有同源L - 甲硫氨酸或非同源L - 高半胱氨酸的三元复合物中也观察到几乎相同的核Overhauser效应模式。距离几何计算使得能够更好地定义和比较每个复合物中允许的腺嘌呤 - 核糖糖苷扭转角的范围和构象空间。对于δMTS.Mg（α，β - 亚甲基）ATP、δMTS.Mg（α，β - 亚甲基）ATP.L - 甲硫氨酸和δMTS.Mg（α，β - 亚甲基）ATP.L - 高半胱氨酸复合物，分别测定了酶结合的Mg（α，β - 亚甲基）ATP的平均腺嘌呤 - 核糖糖苷扭转角为 - 106±9°、 - 99±11°和 - 97±11°。对三种酶结合构象进行比较表明，具有 - 98°腺嘌呤 - 核糖糖苷扭转角且核糖糖环呈3'-内式至O4'-外式褶皱的单一核苷酸结构，在误差范围内与在所有三种复合物中获得的实验核间距离一致。在所有三种复合物中观察到的几乎相同的反式糖苷扭转角表明，酶结合核苷酸的腺苷部分的构象对氨基酰腺苷酸位点结合的氨基酸的存在或性质不敏感。因此，已知在配体结合时甲硫氨酰 - tRNA合成酶中发生的构象变化似乎不会改变核苷酸的结合构象。了解δMTS的氨基酰腺苷酸位点结合的底物的构象和排列信息对于理解氨基酸活化和识别所涉及的分子机制是必要的。
