Interaction of succinate dehydrogenase with succinate and oxaloacetate during enzyme activation-deactivation

The interaction of succinate dehydrogenase from the bovine adrenal cortex with succinate and oxaloacetate was studied in the process of its activation-deactivation. It is supposed that an intermediate unstable complex of succinate dehydrogenase with oxaloacetate plays an important role in the changed enzymic activity.     

The binding site for oxaloacetate on succinate dehydrogenase

Oxaloacetate, a competitive inhibitor of succinate dehydrogenase, bound with a sulfhydryl group of the enzyme to abolish the enzymic activity. Subsequently a thiosemiacetal was apparently formed to render the inhibition practically irreversible. The dehydrogenase, after taking up 25 silver equivalents per flavin, bound little oxaloacetate.     

Tight binding of oxaloacetate to succinate dehydrogenase

[¹⁴C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetone, KCN and ageing at +4° at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored; the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate.     

The activities of the citric acid-cycle enzymes in rat bone-marrow cells

The activities of the eight citric acid-cycle enzymes of rat bone-marrow cells were determined along with several other mitochondrial and non-mitochondrial enzymes. Four of the citric acid-cycle enzymes (aconitase, succinyl-CoA thiokinase, α-oxoglutarate dehydrogenase and succinate dehydrogenase) have closely similar low activities; two [isocitrate dehydrogenase (NAD) and citrate synthase] have intermediate activities; the remaining two (malate dehydrogenase and fumarase) have high activities. The other enzymes surveyed also exhibited a spread of three orders of magnitude, the mitochondrial enzymes showing no less variation than the others.     

The effect of hexokinase and tricarboxylic acid-cycle intermediates on fatty acid oxidation and formation of ketone bodies by rat-liver mitochondria

1. The oxidation of butyrate, hexanoate and octanoate by rat-liver mitochondria suspended in a tris-potassium chloride medium in the presence of malate and serum albumin has been investigated. 2. The oxidation of butyrate to acetoacetate was markedly decreased by the addition of a system competitive for ATP (hexokinase-glucose). 3. Serum albumin or tricarboxylic acid-cycle intermediates prevented the inhibition by hexokinase and in their presence a greater proportion of the oxygen consumption was contributed by the tricarboxylic acid cycle. The results suggest that the energy supply for fatty acid activation is either compartmentalized in a spatial or kinetic sense or there exists a special activating mechanism not involving ATP. 4. Malate and other tricarboxylic acid-cycle intermediates caused substantial reduction (to beta-hydroxybutyrate) of the acetoacetate formed during the oxidation of butyrate, hexanoate and octanoate.     

Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium.

1. The contribution of Co2 fixation to the anaplerotic mechanisms in the myocardium was investigated in isolated perfused rat hearts. 2. K+-induced arrest of the heart was used to elicit a transition in the concentrations of the intermediates of the tricarboxylic acid cycle. 3. Incorporation of 14C from [14]bicarbonate into tricarboxylic acid-cycle intermediates was measured and the rates of the reactions of the cycle were estimated by means of a linear optimization program which solves the differential equations describing a simulation model of the tricarboxylic acid cycle and related reactions. 4. The results showed that the rate of CO2 fixation is dependent on the metabolic state of the myocardium. Upon a sudden diminution of cellular ATP consumption, the pool size of the tricarboxylic acid-cycle metabolites increased and the rate of label incorporation from [14C]bicarbonate into the cycle metabolites increased simultaneously. The computer model was necessary to separate the rapid equilibration between bicarbonate and some metabolites from the potentially anaplerotic reactions. The main route of anaplerosis during metabolite accumulation was through malate + oxaloacetate. Under steady-state conditions there was a constant net outward flow from the tricarboxylic acid cycle via the malate + oxaloacetate pool, with a concomitant anaplerotic flow from metabolites forming succinyl-CoA (3-carboxypropionyl-CoA).     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles

1. Succinate dehydrogenase is inhibited by citrate and β-hydroxybutyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved.
2. Pyruvate, α-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria.
3. Stimulation by α-ketoglutarate and glutamate is not influenced by the presence of rotenone.
4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K⁺ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate.
5. Stimulation by malate is higher in the presence of rotenone.

     

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles.

1. Succinate dehydrogenase is inhibited by citrate and beta-hydroxy-butyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved. 2. Pyruvate, alpha-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria. 3. Stimulation by alpha-ketoglutarate and glutamate is not influenced by the presence of rotenone. 4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K+ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate. 5. Stimulation by malate is higher in the presence of rotenone.     

The role of succinate dehydrogenase and oxaloacetate in metabolic suppression during hibernation and arousal

Hibernation elicits a major reduction in whole-animal O₂ consumption that corresponds with active suppression of liver mitochondrial electron transport capacity at, or downstream of, succinate dehydrogenase (SDH). During arousal from the torpor phase of hibernation this suppression is reversed and metabolic rates rise dramatically. In this study, we used the 13-lined ground squirrel (Ictidomys tridecemlineatus) to assess isolated liver mitochondrial respiration during the torpor phase of hibernation and various stages of arousal to elucidate a potential role of SDH in metabolic suppression. State 3 and state 4 respiration rates were seven- and threefold lower in torpor compared with the summer-active and interbout euthermic states. Respiration rates increased during arousal so that when body temperature reached 30°C in late arousal, state 3 and state 4 respiration were 3.3- and 1.8-fold greater than during torpor, respectively. SDH activity was 72% higher in interbout euthermia than in torpor. Pre-incubating with isocitrate [to alleviate oxaloacetate (OAA) inhibition] increased state 3 respiration rate during torpor by 91%, but this rate was still fourfold lower than that measured in interbout euthermia. Isocitrate pre-incubation also eliminated differences in SDH activity among hibernation bout stages. OAA concentration correlated negatively with both respiration rates and SDH activity. These data suggest that OAA reversibly inhibits SDH in torpor, but cannot fully account for the drastic metabolic suppression observed during this hibernation phase.