Phosphorus-31 nuclear magnetic resonance studies of the binding of nucleotides to NADP+-specific isocitrate dehydrogenase

Abstract

The interaction of the 2''-phosphate-containing nucleotides (NADP, NADPH, 2''-phosphoadenosine 5''-diphosphoribose [Rib-P2-Ado-P], and adenosine 2'',5''-bisphosphate) with NADP-specific isocitrate dehydrogenase was studied by using 31P NMR spectroscopy. The separate resonances corresponding to free and bound nucleotides, characteristic for slow exchange of nuclei on the NMR time scale, were observed in the spectra of the enzyme (obtained in the presence of excess ligand) with NADP and NADPH in the absence and presence of Mg2+ and with Rib-P2-Ado-P in the absence of metal or in the presence of the substrate magnesium isocitrate. The position of the 31P resonance of the bound 2''-phosphate group in these spectra is invariant (.delta. = 6) in the pH range 5-8, indicating that the pK of this group is much lower in the complexes with the enzyme than that (pK = 6.13) in the free nucleotides. The additional downfield shift of this resonance by 1.8 ppm beyond the (.delta. = 4.22) of the dianionic form of the 2''-phosphate in free nucleotides suggests interaction with a positively charged group(s) and/or distortion of P-O-P angles as the result of binding to the enzyme. A single resonance of 2''-phosphate was observed in the spectrum of the enzyme complex with Rib-P2-Ado-P in the presence of Mg2+, with the chemical shift dependent on the nucleotide to enzyme ratio, characteristic for the fast exchange situation. Addition of metal does not perturb the environment of the 2''-phosphate in the complexes of NADP and NADPH with isocitrate dehydrogenase. No detectable enzyme-adenosine 2'',5''-bisphosphate complex was observed under conditions of the NMR experiments. It is postulated that the 2''-phosphate group of NADP, NADPH and Rib-P2-Ado-P binds in a similar environment, presumably to the same site on the enzyme. In contrast to bacterial isocitrate dehydrogenase, both NAD- and NADP-specific enzymes isolated from pig heart do not contain covalently bound phosphate.