Citrate cycle Pyruvate carboxylase

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis see Figure 25.1). /Uternatively, it may be converted directly to 0/ A by pyruvate carboxylase and used in glu-... 

Substrate availability for certain reactions can be optimized by anaplerotic ( topping-up ) reactions. For example, citrate synthase is a key control point of the TCA cycle. The co-substrates of citrate synthase are acetyl-CoA and oxaloacetate (OAA) and clearly, restriction in the availability of either substrate will decrease the rate of the citrate synthase reaction. Suppose, for example, a situation arises when acetyl-CoA concentration is significantly higher than that of OAA, the concentration of the latter can be topped-up and the concentration of acetyl-CoA simultaneously reduced by diverting some of the pyruvate away from acetyl-CoA synthesis (via pyruvate dehydrogenase) to OAA synthesis (via pyruvate carboxylase) as shown in Figure 3.1. The net effect is to balance the relative concentrations of the two co-substrates and thus to promote citrate synthase activity. 

The theory of regulation of the cycle is as follows. First, an increase in oxaloacetate concentration increases the activity of citrate synthase and hence the cycle. The concentration of oxaloacetate is regulated by the activity of the enzyme pyruvate carboxylase, which catalyses the reaction ... 

Pyruvate carboxylase is a regulatory enzyme and is virtually inactive in the absence of acetyl-CoA, its positive allosteric modulator. Whenever acetyl-CoA, the fuel for the citric acid cycle, is present in excess, it stimulates the pyruvate carboxylase reaction to produce more oxaloacetate, enabling the cycle to use more acetyl-CoA in the citrate synthase reaction. 

ADP, acetyl-CoA, succinyl-CoA, and citrate. The major known sites for regulation of the cycle include two enzymes outside the cycle (pyruvate dehydrogenase and pyruvate carboxylase) and three enzymes inside the cycle (citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase). All of these sites of regulation represent important metabolic branchpoints. 

Answer Anaplerotic reactions replenish intermediates in the citric acid cycle. Net synthesis of a-ketoglutarate from pyruvate occurs by the sequential actions of (1) pyruvate carboxylase (which makes extra molecules of oxaloacetate), (2) pyruvate dehydrogenase, and the citric acid cycle enzymes (3) citrate synthase, (4) aconitase, and (5) isocitrate dehydrogenase ... 

Answer Fatty acid catabolism increases the level of acetyl-CoA, which stimulates pyruvate carboxylase. The resulting increase in oxaloacetate concentration stimulates acetyl-CoA consumption through the citric acid cycle, causing the citrate and ATP concentrations to rise. These metabolites inhibit glycolysis at PFK-1 and inhibit pyruvate dehydrogenase, effectively slowing the utilization of sugars and pyruvate. 

The product of this reaction, oxaloacetate, can either enter the gluconeogenic pathway (Chap. 11) by way of malate or condense with acetyl-CoA to yield citrate. Pyruvate carboxylase is an allosteric enzyme, and it is activated by the heterotropic effector, acetyl-CoA. Thus, pyruvate in the mitochondria is the substrate for either pyruvate dehydrogenase or pyruvate carboxylase, the activities of which, in turn, are controlled by reactants associated with the citric acid cycle. The interplay among pyruvate dehydrogenase, pyruvate carboxylase, pyruvate, and the citric acid cycle is shown in Fig. 12-9. 

The cycle oxidizes acetyl-CoA, and to perform this task, it must convert acetyl-CoA to citrate. For this to be achieved, oxaloacetate must be available. If the removal of intermediates results in a decrease in the amount of oxaloacetate for this purpose, acetyl-CoA cannot be removed and will accumulate. This will inhibit the pyruvate dehydrogenase complex and activate pyruvate carboxylase, leading to the conversion of pyruvate to oxaloacetate. This product is now available to condense with the acetyl-CoA to produce citrate, which will restore the status quo. Reactions like that of pyruvate carboxylase that provide molecules for the replacement of intermediates of the citric acid cycle are known as anaplerotic reactions (Greek, meaning to fill up ana = up + plerotikos from pleroun = to make full ). 

Pyruvate can be converted to acetyl-CoA via the pyruvate dehydrogenase complex. Pyruvate can also be carboxylated via pyruvate carboxylase to produce oxaloacetate. So, two molecules of pyruvate can form the precursors of citrate, which can be converted to succinate within the citric acid cycle. 

Genetic deficiency of pyruvate carboxylase does not cause the expected hypoglycemia. Rather, it seems that depletion of tissue pools of oxaloacetate results in impaired activity of citrate synthase, and a slowing of citric acid cycle activity, leading to accumulation of lactate, pyruvate, and alanine, and also increased accumulation of acetyl CoA, resulting in ketosis. Affected infants have serious neurological problems and rarely survive. A less severe variant of the disease is associated with low residual activity of pyruvate carboxylase. 

The acetyl CoA formed in fatty acid oxidation enters the citric acid cycle only if fat and carbohydrate degradation are appropriately balanced. The reason is that the entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate, but the concentration of oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. Recall that oxaloacetate is normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase (Section 16.3.1). This is the molecular basis of the adage that fats burn in the flame of carbohydrates. 

Pyruvate carboxylase is activated by acetyl CoA and inhibited by high concentrations of many acyl CoA derivatives. As the concentration of oxaloacetate is depleted through the efflux of TCA cycle intermediates, the rate of the citrate synthase reaction decreases and acetyl CoA concentration rises. The acetyl CoA then activates pyruvate carboxylase to synthesize more oxaloacetate. 

Pyruvate is also converted to oxaloacetate. The enzyme that catalyzes this reaction, pyruvate carboxylase, is activated by acetyl CoA. Because acetyl CoA cannot directly cross the mitochondrial membrane to form fatty acids in the cytosol, it condenses with oxaloacetate, producing citrate. The citrate that is not required for tricarboxylic acid (TCA) cycle activity crosses the membrane and enters the cytosol. 

In addition, it has become increasingly evident that there is significant mitochondrial dysfunction and impairment of the oxidative phosphorylation system [29, 41, 66-69]. This impairment is felt to be secondary to inhibition of the Krebs cycle enzymes citrate synthase, aconitase, and isocitrate dehydrogenase by methylcitrate, inhibition of pyruvate carboxylase by methylmalonic acid, and inhibition of pyruvate dehydrogenase complex. 

Rapid p-oxidation of fatty acids in perfused liver (DeBeer et a/., 1974) and in isolated mitochondria (Lopes-Cardozo and Van den Bergh, 1972) has been shown to suppress the operation of citric acid cycle apparently from the elevation of mitochondrial NADH/NAD ratio which restricts oxaloaceta-te availability for citrate synthase and simultaneously inhibits isocitrate oxidation (Lenartowicz et a/., 1976). Considerable support for an earlier postulate that oxaloacetate availability normally determines the rate of citrate synthesis has become available. Thus, because of marked protein binding, the concentration of free, as opposed to total, oxaloacetate in matrix of liver mitochondria is now estimated to be near the of citrate synthase (Siess et al., 1976 Brocks eta ., 1980). The antiketogenic effect of alanine (Nosadini et a/., 1980) and of 3-mercaptopicolinate, an inhibitor of phosphoenolpy-ruvate carboxykinase (Blackshear et a/., 1975), is believed to be exerted, at least in part, from their ability to raise hepatic oxaloacetate concentration. And, in pyruvate carboxylase deficiency, expected to impair oxaloacetate supply, concentration of ketone bodies is elevated (Saudubray et a/., 1976). 

A further example is the enzyme pyruvate carboxylase which is involved both in gluooneo-genesis and in maintaining the level of citrate cycle intermediates and requires acetyl-CoA as an obligatory activator. 

Pyruvate carboxylase is a mitochondrial enzyme which requires Mg and biotin as a carrier of activated carbon dioxide. In addition, it has an absolute requirement for acetyl-CoA, which acts as an allosteric activator (page 340). In this way the accumulation of acetyl-CoA normally triggers the formation of oxaloacetate. If ATP is in short supply, oxaloacetate will combine with some of the acetyl-CoA to form citrate, and so be drawn into the citrate cycle. On the other hand, if there is a surplus of ATP, both this and the oxaloacetate will be used for gluconeogenesis. 

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