Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria

Abstract

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.

Quantifying Reductive Carboxylation Flux of Glutamine to Lipid in a Brown Adipocyte Cell Line

“…On days 2 and 4, the medium was replaced with fresh differentiation medium. To estimate metabolic fluxes, 13 C-labeled glutamine and glucose were substituted for the unlabeled metabolites in the medium as described for specific experiments. To evaluate the effect of inhibitors of IDP, the differentiation medium was supplemented with oxalomalate or 2-methylisocitrate as indicated and incubated at 37°C for 10 min, followed by addition of 13 C-labeled substrates.…”

“…De novo fatty acid synthesis, using glutamine as carbon source, may occur by either of two pathways: glutaminolysis, which converts C3 and C4 of glutamine to acetyl-CoA, and reductive carboxylation, which converts C4 and C5 of glutamine to acetyl-CoA. To evaluate the relative fluxes from glutamine to lipid via reductive carboxylation and glutaminolysis we compared the contribution of [5-13 C]glutamine and [U- 13 C]glutamine to lipogenic acetyl-CoA using ISA. ISA estimated that [U- 13 C]glutamine contributed 38 -40% of the lipogenic acetyl-CoA.…”

“…To evaluate the relative fluxes from glutamine to lipid via reductive carboxylation and glutaminolysis we compared the contribution of [5-13 C]glutamine and [U- 13 C]glutamine to lipogenic acetyl-CoA using ISA. ISA estimated that [U- 13 C]glutamine contributed 38 -40% of the lipogenic acetyl-CoA. The ISA D value for [U- 13 C]glutamine represents the sum of fluxes through the two pathways.…”

“…Early studies established the feasibility of reductive carboxylation flux through this enzyme in hepatic models (11,12). Flux of NADP-dependent enzyme toward ␣-ketoglutarate in vivo is supported by the affinity constant of the enzyme for CO 2 (K m ϳ 1.6 mM), which is very close to physiological CO 2 concentration (1.5 mM) and the affinity of the enzyme for NADPH, which is 100-fold higher than for NADP ϩ (13). Additionally, reductive carboxylation flux through IDPm has been hypothesized to play a key role in metabolic regulation of CAC cycle flux via a substrate cycle involving NAD/NADP transhydrogenase located at the inner mitochondrial membrane (14).…”

“…An alternative to glutaminolysis is the reductive carboxylation pathway where glutamine enters the CAC as ␣-ketoglutarate and is converted to citrate via isocitrate dehydrogenase (IDH) operating in reverse of the CAC direction. Evidence for reductive carboxylation in transformed cells is the finding that [5][6][7][8][9][10][11][12][13][14] C]glutamine labels acetyl-CoA-derived carbons of lipids (6,7). Additionally, reversal of IDH has been detected in perfused liver and heart as evidenced by the mass isotopomer distribution (MID) of [ 13 C]citrate following perfusion with [ 13 C]glutamine (8,9).…”