1H-NMR investigation of the interaction between RNase T1 and a novel substrate analog, 2'-deoxy-2'-fluoroguanylyl-(3'-5')uridine.
The interaction between RNase T1 and a non-hydrolysable substrate analog, 2'-deoxy-2'-fluoroguanylyl-(3'-5')uridine (GfpU), was investigated using 1H-NMR spectroscopy. In the complex, the Gfp 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 (Gfp). However, in contrast to the cases of these two inhibitors, the complex formation with GfpU 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 (pKa 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.
Shibata
Y
Y
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
Shimada
I
I
Ikehara
M
M
Miyazawa
T
T
Inagaki
F
F
D001230:Aspergillus / Q000201:enzymology*; D001236:Aspergillus oryzae / Q000201:enzymology*; D015226:Dinucleoside Phosphates; D004722:Endoribonucleases / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
D015226:Dinucleoside Phosphates; D006150:Guanine Nucleotides; D006639:Histidine; C056777:2'-deoxy-2'-fluoroguanylyl-(3'-5')uridine; D006157:Guanosine Monophosphate; D004722:Endoribonucleases; D006163:Ribonuclease T1; D014529:Uridine