Binding of ligands to half of subunits of NAD-dependent isocitrate dehydrogenase from pig heart. Binding of manganous ion, isocitrate, ADP and NAD.

NAD-dependent isocitrate dehydrogenase from pig heart contains three types of subunits of slightly different molecular weights in an approximate ratio of 2:l:l. Ultrafiltration binding experiments at pH 6.1 indicate 1 binding site for every 2 subunits for several different ligands. The metal activator, Mn2+, binds to isocitrate dehydrogenase in the absence of other ligands ( K D = 115 p ~ ) . Mn2+ binding at the 1 site/2 subunits is tightened by addition of the enzyme regulator, ADP. Direct measurement of ADP binding reveals 1 high affinity metal-dependent ADP site per 2 subunits; however, when free metal is chelated by EDTA, ADP binds only weakly. Analysis of the ADP concentration dependence (KD = 4.8 p ~ ) of manganese binding and the manganese concentration dependence of ADP3binding (KO = 2.6 p ~ ) indicates that free metal ion and free ADP bind independently to the enzyme. ADP binding is not promoted by calcium ion. In the presence of Mn2+, ADP binding is correlated with enzyme aggregation as measured by sedimentation velocity. Isocitrate also binds weakly when free metal is chelated by EDTA. The binding of both MnZ+ and isocitrate appears tighter when added together and there is 1 metal-dependent isocitrate site per 2 subunits. Addition of ADP with these ligands tightens manganese binding by about 2-fold and reduces the dissociation constant for free dibasic isocitrate from 21 to 3.4 p ~ . Manganese and isocitrate binding data obtained in the presence of ADP are most consistent with both ligands binding independently. The enhancement of isocitrate binding by ADP is also obtained with magnesium ion as the metal activator, but no requirement for calcium ion is observed. NAD+, with or without manganous ion, also binds to 1 site/% subunits with a dissociation constant of 55 p ~ , which is close to the Michaelis constant and consistent with an ordered mechanism in which NAD+ binds prior to isocitrate. The stoichiometry of ligand binding suggests either that isocitrate dehydrogenase has half the number of catalytic and ADP regulatory sites as it has subunits or that strong egative cooperativity exists in ligand binding.