title: Interrelationships among nucleotide binding sites of pig heart NAD-dependent isocitrate dehydrogenase.
abstract: Binding of NADH and NADPH to NAD-linked isocitrate dehydrogenase from pig heart (which contains three types of subunits of similar molecular weight in the ratio 2:1:1) was studied by use of the enhancement of nucleotide fluorescence. NADH and NADPH bind independently and, for both reduced nucleotides, 0.5 binding sites/average subunit or two binding sites for every four subunits were detected. Binding of NADH is unaffected by metal ion, isocitrate, or NAD+. ADP is also not competitive with NADH binding but reduces the strength of binding 3-fold. In contrast, ATP (KI = 9 microM ATP4-) appears to be competitive with NADH. Evidence for an NADH site with higher dissociation constant (KD greater than 200 microM) than that obtained from the fluorescence measurements (KD = 2.8 microM at pH 6.1) is indicated by competition of NADH with [14C]NAD+ binding. NADPH binding is enhanced by the presence of manganese. The metal dependence is consistent with NADPH (KD = 8.1 microM at pH 6) binding to the same site as Mn-NADPH2- (KD = 0.9 microM). Dissociation constants for both species increase with increasing pH in the range 6-8. NADPH binding is competitively inhibited by ADP, ATP, ADP-ribose, and isocitrate. This inhibition of NADPH binding is probably indirect since measurements of [14C]ADP in the presence and absence of NADPH showed little inhibition of binding by NADPH concentrations sufficient to saturate the fluorescent site. These studies extent the number of ligands for isocitrate dehydrogenase for which there are fewer binding sites than there are subunits.
mesh_terms: D000818:Animals; D001665:Binding Sites; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013552:Swine
chemical_list: D009243:NAD; D008345:Manganese; D009249:NADP; D007521:Isocitrate Dehydrogenase