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  • Title: 1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase.
    Author: Ehrlich RS, Colman RF.
    Journal: Biochemistry; 1985 Sep 24; 24(20):5378-87. PubMed ID: 4074702.
    Abstract:
    The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2',5'-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide 1'-ribose proton and the 2-nicotinamide ring proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine 1'-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn adenine--ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nicotinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from less than 7 s-1 for NADPH to approximately 1600 s-1 for adenosine 2',5'-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2',5'-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The 1H NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between delta 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK = 5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at delta 7.55 indicates proximity of either a nontitrating histidine or an aromatic residue to the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that the adenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein--nucleotide interactions. The alterations in enzyme--nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.
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