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1 H‐NMR investigation of the interaction between RNase T 1 and a novel substrate analog, 2′‐deoxy‐2′‐fluoroguanylyl‐(3′–5′)uridine
Author(s) -
Shibata Yasuyuki,
Shimada Ichio,
Ikehara Morio,
Miyazawa Tatsuo,
Inagaki Fuyuhiko
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)81270-5
Subject(s) - chemistry , rnase p , stereochemistry , moiety , protonation , glycosidic bond , nuclear magnetic resonance spectroscopy , histidine , ribose , substrate (aquarium) , residue (chemistry) , rnase ph , active site , ribonuclease t1 , uridine , enzyme , rna , biochemistry , organic chemistry , ion , oceanography , gene , geology
The interaction between RNase T 1 and a non‐hydrolysable substrate analog, 2′‐deoxy‐2′‐fluoroguanylyl‐(3′–5′)uridine (G fp U), was investigated using 1 H‐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 fp U at pH 6.0 was found to shift the His‐40 C2 proton resonance of RNase T 1 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 (p K 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.