Open AccessComplexes of Ribonuclease A with 2′‐Deoxy‐2′‐Fluororibose Substrate Analogues Studied by Nuclear Magnetic Resonance
Author(s) -
ANTONOV Ilja V.,
GUREVICH Aleksander Z.,
DUDKIN Sergey M.,
KARPEISKY Marat Ya.,
SAKHAROVSKY Valentin G.,
YAKOVLEV Gennady I.
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12350.x
Subject(s) - chemistry , ribonuclease t1 , glycosidic bond , protonation , histidine , stereochemistry , phosphodiester bond , imidazole , intramolecular force , crystallography , ligand (biochemistry) , alkane stereochemistry , chemical shift , ribose , nuclear overhauser effect , nuclear magnetic resonance spectroscopy , nuclear magnetic resonance , enzyme , rnase p , rna , crystal structure , ion , biochemistry , receptor , physics , organic chemistry , gene
Chemical shifts of the histidine C‐2 protons, the Tyr‐25 protons in RNAase A and the 2′‐deoxy‐2′‐fluorouridilyl(3′‐5′)adenosine (2′‐FDUrd‐ P ‐Ado) protons were measured as a function of the concentration of the ligand bound to the enzyme at pH 5.5 in order to determine the position and mutual orientation of the dinucleoside monophosphate bases in the complex. It is concluded that in its complex with RNAase 2′‐FDUrd‐ P Ado is fixed at the active site in an extended conformation precluding intramolecular interaction of the bases. Nuclear Overhauser effect measurements for the H‐1′ ribose protons and the base protons (H‐6, H‐8) were made in order to determine glycoside torsion angles in both fragments of the dinucleoside monophosphate in the complex. Comparison of the experimental results with theoretical values for the dependence of nuclear Overhaused effect on the glycosidic torsion angle for the protons studied indicates that both fragments in 2′‐FDUrd‐ P ‐Ado have the anti conformation in the complex. The pH dependence of the chemical shifts for the active‐site histidine protons and for the protons of the Tyr‐25 aromatic ring of the complex was srudied in the ph range of 3.0–9.2. The results suggest that there is a direct interaction between the negatively charged phosphodiester fragment of a ligand the protonated imidazole ring of His‐119 in the complex. This conclusion is supported by the pH dependence of the chemical shift for 31p of 2′‐FDUrd‐ P ‐Ado in the complex with RNAase A. The chemical shift for the Tyr‐25 protons in the complexes of RNAase A with nucleotide ligands was shown to depend upon the ionization state of His‐119 and a group in the protein with a p K 3.6. A model for the structure of the Michaelis complex formed by RNAase with substrate is proposed.