| The TPN-linked isocitrate dehydrogenase from pig heart has been purified to homogeneity using Blue Sepharose Cl-6B chromatography as the only column chromatographic step and subsequently crystallized. Molecular sieve chromatography and analytical ultracentrifugation showed that the enzyme dimerized in response to magnesium DL-isocitrate. TPNH or EDTA also brought about this effect whereas TPN('+) did not. The molecular weight of the monomer was 60,000 (+OR-) 2,500 with an S(,20,w) of 4.8 (+OR-) 0.1 and a Stokes radius of 29 (ANGSTROM). The molecular weight of the dimer was 115,000 (+OR-) 5,000 with and S(,20,w) of 6.7 (+OR-) 0.2 and a Stokes radius of 39 (ANGSTROM). Active enzyme centrifugation showed that the dimer was the active species in the oxidative decarboxylation of isocitrate.;In double inhibitor experiments with the reaction in the direction of reductive carboxylation of (alpha)-ketoglutarate, the results were consistent with mutual facilitation of the binding to monomer of (alpha)-methylisocitrate and TPN('+). When the dimeric form was prevalent the specific activity of the enzyme was maximal, the order of addition of substrates was random, and, in accord with previous studies by Uhr et al. (1974) (J. Biol. Chem. 249, 2920-2927) inhibition by (alpha)-methylisocitrate was competitive with isocitrate (K(,is) = 0.2 (mu)M) and noncompetitive with TPN('+). This monomer-dimer equilibrium is dynamic and responsive to conditions which affect this equilibrium. A model has been proposed in which the rate limiting step in the ordered rapid equilibrium mode is determined by the rate of conversion of the substrate bound monomer form to the corresponding dimer form before catalysis ensues. The steady state equilibrium position of the free monomer and dimer forms determines the overall rate of catalysis and the kinetic mechanism. In confirmation of this model, at low enzyme concentrations (monomer prevalent) the addition of a ligand favoring displacement of the monomer-dimer equilibrium toward dimer (e.g. magnesium isocitrate, TPNH, EDTA) enhanced the rate of catalysis, shifted the mechanism from the ordered rapid equilibrium to the random mode and eliminated the tight binding inhibition by (alpha)-methylisocitrate.;In agreement with the results of these physicochemical studies, the kinetic properties of crystalline TPN-isocitrate dehydrogenase correspond to those of a dissociating dimeric enzyme. Investigations of the enzyme by the Kurganov dilution approach (B.I. Kurganov (1967) Molek. Biol. (Moscow) 1, 17-27), revealed that the dimeric enzyme was active and the monomer was inactive. These results with TPN-isocitrate dehydrogenase from pig heart concur with those reported for the enzyme from bovine liver (M.F. Carlier and D. Pantaloni (1978) Eur. J. Biochem. 89, 571-578). Under conditions where the monomeric form was predominant (e.g. at low enzyme concentration) the specific activity declined and dimerization appeared to be the rate limiting step in the reaction. Under these conditions, the enzyme exhibited an ordered rapid equilibrium mechanism with magnesium isocitrate adding before TPN('+) and under pre-steady state conditions, inhibited by DL-threo-(alpha)-methylisocitrate was of the tight binding type, with a K(,i) approaching zero at saturating TPN.('+). |
