Binding of vanadate (V) to ribonuclease-T1 and inosine, investigated by 51V NMR spectroscopy.
Ribonuclease T1 (RNase-T1) from Aspergillus Oryzae cleaves ribonucleic acid specifically at guanosine to yield oligonucleotides with terminal guanosine-3'-phosphate. It forms a complex with vanadate (association constant K approximately 145 +/- 30 M-1; delta (51V) = -514 ppm) with spectral features similar to the less stable complexes obtained with di- and tripeptides (Gly-His, Pros-His-Ala, Gly-His-Lys, Val-Glu) containing amino acids that are constituents at the active site of the enzyme. Guanosine also forms a (sparingly soluble) complex with vanadate. Its role is mimicked by inosine, which yields two soluble complexes with vanadate, characterized by delta values of -511 (K = 94 M-1) and -523 ppm (K = 305 M-1 in TRIS buffer and 685 m-1 in buffer-free solution). Comparison with literature values leads to an assignment of the delta = -523 signal to a complex where monovanadate, possibly in a trigonal bipyramidal geometry suggested for the transition state of the phosphate analogue, is coordinated to the 2'- and 3'-oxygens of the ribose ring. A drastic increase of complex stability is observed in the ternary vanadate (12-16 mM)/inosine(10.5 mM)/RNase-T1(5.4 mM) system. An approximate lower limit for the association constant is 1.5.10(5) M-2. The spectral characteristics of the main component of the binary vanadate/inosine complex are essentially maintained (delta = -525 ppm, half-width = 960 Hz), suggesting vanadate binding to the enzyme through hydrogen bonds.
Rehder
D
D
Institut für Anorganische und Angewandte Chemie, Universität Hamburg, FRG.
Holst
H
H
Quaas
R
R
Hinrichs
W
W
Hahn
U
U
Saenger
W
W
D004722:Endoribonucleases / Q000378:metabolism*; D007288:Inosine / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D006163:Ribonuclease T1 / Q000378:metabolism*; D014638:Vanadates
D010455:Peptides; D014638:Vanadates; D007288:Inosine; D004722:Endoribonucleases; D006163:Ribonuclease T1