On the function of N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine in transfer ribonucleic acid. Metal ion binding studies.

The unusual transfer ribonucleic acid (RNA) anticodon adjacent modified nucleoside N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine, t6A, and its N6-methyl (mt6A) and 5'-phosphate (pt6A) derivatives are efficient ligands for magnesium and manganese ions, as demonstrated by potentiometry and nuclear magnetic resonance spectroscopy. Analysis of the 1H and 13C NMR spectra of t6A in the presence of paramagnetic Mn (II) at 32-34 degrees C has shown that the metal ion binds to the carboxyl group and probably N6 of the side chain as well as the N1 or N7 atom of the adenine ring. A more specific and stronger metal-ligand complex between Mn(II) and pt6A is evident from the 1H, 13C, and 31P NMR data. In this case, the metal forms a complex involving phosphate, N7 of the adenine and the carboxyl group, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparisup, and N6 of the side chain. Blocking the N6 site as in mt6A attenuates the interaction, as revealed in the proton spectra. Potentiometric titrations at 30 degree C and in 0.1 M KNO3 have produced findings parallel to the NMR data on the interaction of Mn(II) with these ligands and have allowed a quantitative comparison between them as well as a comparison of the binding between Mg(II) and Mn(II). The stability constants (log K) for 1:1 metal-ligand complexes between Mg(II) and t6A, mt6A, and pt6A are respectively 5.5, 4.3, and 7.1. For Mn(II), the respective values are 6.0, 4.5, and 7.9. In the case of pt6A, the stability constants are about 5 log K units larger than those obtained for Mg(II) and Mn(II) binding to 5'-AMP [Khan, M. M. T., & Martell, A. E. (1967) J. Am. Chem. Soc. 89, 5585]. Thus the threonine side chain is an important determinant in the interaction between the modified nucleosides and metal ions, and these results are supportive of the idea that a facet of the function of this type of unusual nucleoside in transfer RNA is as a specific ligand for magnesium ion, a postulate promulgated earlier [Miller, J. P., Hussain, Z., & Schweizer, M. P. (1976) Nucleic Acids Res. 3, 1185].