Phosphorus-31 nuclear magnetic resonance studies of the methylene and fluoro analogues of adenine nucleotides. Effects of pH and magnesium ion binding.

Abstract

The /sup 31/P NMR spectra of methylene and fluoro analogues of adenine nucleotides have been studied as a function of pH and Mg/sup 2 +/ concentration. As expected, the spectra of the fluoro analogues are not a function of pH. Mg/sup 2 +/ titrations show that both fluoro analogues bind the metal ion less strongly than ATP or ADP, respectively. For the analogues AMPPCP (..beta..,..gamma..) and AMPCP (..cap alpha..,..beta..), a pK/sub a/ near 8.1 was deduced for the titration of the terminal phosphoryl group; in the presence of Mg/sup 2 +/ this value is lowered to about 6.3. The other ATP analogue, AMPCPP (..cap alpha..,..beta..), has a titration behavior virtually identical with that of ATP. Therefore, at physiological pH all adenine nucleotides will be fully deprotonated in the presence of Mg/sup 2 +/ ions. A comparison of the magnitudes and directions of the chemical shift changes observed due to protonation leads to the conclusion that these are caused by changes in the structure of the polyphosphate chain. Mutual titrations indicate that both methylene ATP analogues can bind one Mg/sup 2 +/ tightly and a second ion more weakly. The methylene analogue of ADP binds only one Mg/sup 2 +/. Thesemore » data show that the methylene analogues compare favorably with ATP and ADP with respect to metal binding properties. A comparison of the results with those reported for imido- and thioadenine analogues suggests that the /sup 31/P NMR spectra for metal complexed nucleotides fee in solution are probably the fact-exchange average of all metal-coordination structures possible. This raises questions about the validity of direct comparisons of these spectra to those observed for enzyme-bound nucleotides. (JMT)« less