Regulation of Mitochondrial α-Ketoglutarate Metabolism by Product Inhibition at α-Ketoglutarate Dehydrogenase

The mechanism for regulation of α-ketoglutarate metabolism by the phosphorylation state of the intramitochondrial nucleotides has been investigated using intact rat and guinea pig liver mitochondria and isolated α-ketoglutarate dehydrogenase. Depletion of ATP under conditions of either high or low intramitochondrial NADH levels was achieved by addition of oligomycin plus ADP and glucose-hexokinase as an extramitochondrial ATP trapping system to State 4 or uncoupled mitochondria, or by a stimulation of the rate of GTP utilization for P-enolpyruvate synthesis by malate addition. A decrease of the ATP:ADP ratio in rat liver mitochondria resulted in a decrease of the succinyl-CoA to CoA-SH ratio. The GTP:GDP ratio, calculated by assuming equilibrium at succinate thiokinase, also decreased over a 4-fold range but the measured GTP content (approximately 0.2 nmoles per mg of protein) remained constant within analytical error. Nucleoside diphosphate kinase was calculated to be displaced from equilibrium by about two orders of magnitude. Flux through α-ketoglutarate dehydrogenase, as measured by α-ketoglutarate uptake, varied inversely with the succinyl-CoA to CoA-SH ratio at both high and low levels of intramitochondrial NADH. The rate of α-ketoglutarate uptake by mitochondria having approximately equal intramitochondrial phosphate potentials and succinyl-CoA to CoA-SH ratios was greater at low than at high ratios of NADH:NAD. Addition of malate to guinea pig liver mitochondria incubated with α-ketoglutarate in the presence of uncoupler and oligomycin caused a 2-fold stimulation of α-ketoglutarate uptake, a 4.4-fold increase of P-enolpyruvate production and a 4-fold decrease of the succinyl-CoA to CoA-SH ratio. The ATP content decreased from 3.5 to 1.1 nmoles per mg of protein while the GTP content changed insignificantly from 0.21 to 0.18 nmoles per mg of protein. With guinea pig liver mitochondria at low NADH levels, a linear relationship was obtained between the reciprocal of the α-ketoglutarate uptake and the succinyl-CoA to CoA-SH ratio, which provided a value of 1.6 for the ratio of Michaelis constant for CoA-SH to the inhibition constant for succinylCoA. Initial velocity kinetics with purified pig heart α-ketoglutarate dehydrogenase showed that succinyl-CoA and NADH were strong inhibitors, but no inhibitory effects were obtained with GTP or ATP. The inhibition by succinyl-CoA was independent of the NADH:NAD ratio and was competitive with CoA-SH. At high α-ketoglutarate and NAD concentrations, the Km for CoA-SH was 2.7 µm and the Ki for succinyl-CoA was 6.9 µm. Inhibition by NADH was noncompetitive with respect to both NAD and α-ketoglutarate. At high α-ketoglutarate and CoA-SH concentrations, the Km for NAD was 21 µm and the Ki for NADH was 4.5 µm. It is concluded that regulation of flux through α-ketoglutarate dehydrogenase by the state of phosphorylation of the nucleotide systems in rat and guinea pig liver mitochondria is achieved primarily by variations of the succinyl-CoA to CoA-SH ratio and to a lesser extent by variations of the NAD oxidation-reduction state. There are no grounds for supposing a direct effect via GTP or ATP.