Binding of vanadate (V) to ribonuclease-T 1 and inosine, investigated by 15V NMR spectroscopy

Abstract

Ribobuclease T 1 ( RNase-T 1 ) 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 ≈ 145 ± 30 M −1; δ( 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 δ 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 δ = −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-T 1(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 (δ = −525 ppm, half-width = 960 Hz), suggesting vanadate binding to the enzyme through hydrogen bonds.