Supramolecular organization of tricarboxylic acid cycle enzymes

We propose a spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon). The structure is based on an analysis of data on the interaction between tricarboxylic acid cycle enzymes and the mitochondrial inner membrane, as well as on data on enzyme-enzyme interactions. The alpha-ketoglutarate dehydrogenase complex, adsorbed along one of the 3-fold symmetry axes of the mitochondrial inner membrane, plays a key role in formation of the metabolon. In the interaction with the membrane, two association sites of the alpha-ketoglutarate dehydrogenase complex participate, placed on opposite sides of the complex. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleoside-diphosphate kinase. Succinate dehydrogenase, which is the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of the metabolon on the membrane. The molecular mass of the complex (without regard to succinate dehydrogenase) is 8 x 10(6) Da. The metabolon symmetry corresponds to the D3 point symmetry group.     

Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria

Sonic oscillation of mitochondria usually leads to the release of a number of Krebs tricarboxylic acid cycle enzymes. These enzymes have, therefore, been referred to as soluble matrix enzymes. In the present report, we show that gentle sonic or osmotic disruption can be used to obtain a mitochondrial preparation where these enzymes appear to be organized in a large complex of proteins. Using citrate synthase as a marker for these enzymes, we show that the proposed complex is easily sedimented at 32,000 X g in 30 min. The exposed citrate synthase in these complexes can be inhibited by its antibody, indicating that the enzymes are not merely entrapped in substrate-permeable vesicles. The effects of pH, temperature, ionic strength, and several metabolites on the ability to obtain the sedimentable citrate synthase have been tested. These studies indicate that the complex is stable at conditions presumed to exist in situ. Electron microscopic studies show that gentle sonic oscillation gives rise to an efflux of mitochondrial matrix contents which tend to remain attached to the original membranes. The sedimentable fraction also contained four other presumably soluble Krebs tricarboxylic acid cycle enzymes: aconitase, NAD+-isocitrate dehydrogenase, fumarase, and malate dehydrogenase.     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.     

Thermostability of enzymes of the tricarboxylic acid cycle ofBacillus coagulans

The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile,Bacillus coagulans. Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30°C compared with that in cultures grown at 55°C, this difference could be largely eliminated by the removal of cell-wall material. The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55°C than in those grown at 30°C. The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature. Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase. Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates. These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.     

Incomplete tricarboxylic acid cycle in a type I methylotroph, Methylococcus capsulatus.

Alpha-Ketoglutaratedehydrogenase was undetectable in extracts of Methylococcus capsulatus. Cells incorporated [1-14-C] acetate into only four protein amino acids (glutamate, proline, arginine, and leucine) and the C5, but not C1, of glutamate.     

Stoichiometric traps in the tricarboxylic acid cycle. I. Self-inhibition and triggering phenomena

The steady-state oxidation of 2 mM pyruvate in pigeon and rat heart mitochondria in the presence of ADP-glucose-hexokinase load can be strongly inhibited by excess (10-40 mM) of pyruvate or beta-hydroxybutyrate. This inhibition is accompanied by the accumulation of alpha-ketoglutarate and a decrease of malate. The mechanism of such substrate inhibition may be associated with the limitation of the tricarboxylic acid cycle flux by low levels of oxaloacetate and free CoA due to their being trapped as alpha-ketoglutarate and acetyl-CoA. Contrary to pyruvate, the ketone bodies in the absence of other substrates produce self-inhibition of their oxidation at as low concentrations as 0.5-1 mM. At 10-15 mM of acetoacetate, a complete suppression of respiration may develop. At a high load (preset by ADP or the uncoupler CCCP), the suppression is characterised by the accumulation of malate and a decrease of alpha-ketoglutarate. At low loads, the reverse distribution of the intermediates takes place. It is concluded that the system of ketone body oxidation in heart mitochondria is an example of biochemical triggers (systems with two alternative stable states).