1H‐NMR investigation of the interaction between RNase T1 and a novel substrate analog, 2′‐deoxy‐2′‐fluoroguanylyl‐(3′–5′)uridine

FEBS Letters

Miyazawa, Tatsuo³  Shibata, Yasuyuki³  Ikehara, Morio¹  Shimada, Ichio²  Inagaki, Fuyuhiko² 

[1] Institute of Protein Engineering, Nipponbashi 103, Tokyo Japan;The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan;Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

关键词: NMR;    RNase T1;    2′-Deoxy-2′-fluoroguanylyl-(3′–5′)uridine;    Nuclear Overhauser effect;    GfpU;    2′-deoxy-2′-fluoroguanylyl-(3′–5′)uridine;    NOE;    nuclear Overhauser effect;    Gfp;    2′-deoxy-2′-fluoroguanosine 3′-monophosphate;    DSS;    sodium 2;    2-dimethyl-2-silapentane-5-sulfonate;   

DOI  :  10.1016/0014-5793(88)81270-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The interaction between RNase T₁ and a non-hydrolysable substrate analog, 2′-deoxy-2′-fluoroguanylyl-(3′–5′)uridine (G_fpU), was investigated using ¹H-NMR spectroscopy. In the complex, the G_fp portion takes the syn form around the glycosidic bond and the 3′-endo form for the ribose moiety, similar to those found in 3′-GMP and 2′-deoxy-2′-fluoroguanosine 3′-monophosphate (G_fp). However, in contrast to the cases of these two inhibitors, the complex formation with G_fpU at pH 6.0 was found to shift the His-40 C2 proton resonance of RNase T₁ to high field as much as 1 ppm. At pH 6.0, this histidine residue appears to be unprotonated in the complex, but is protonated in the free enzyme (pK _a of His-40 being 7.9). His-40, rather than Glu-58, is probably involved in the catalytic mechanism as a Lewis base, supporting the recent results from site-directed mutagenesis.

【 授权许可】

Unknown