The citric acid cycle is not only a pathway for oxidation of two carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids (see Chapters 28 and 29), and providing the substrates for amino acid synthesis by transamination (see Chapter 27), as well as for gluconeogenesis (see Chapter 19) and fatty acid synthesis (see Chapter 23). Because it functions in both oxidative and synthetic processes, it is amphibolic (Figure 16–4).
Involvement of the citric acid cycle in transamination and gluconeogenesis. The bold arrows indicate the main pathway of gluconeogenesis.
The Citric Acid Cycle Takes Part in Gluconeogenesis, Transamination, & Deamination
All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence net production of glucose (in the liver and kidney, the organs that carry out gluconeogenesis; see Chapter 19). The key enzyme that catalyzes net transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor (see Figure 19–1). The GTP required for this reaction is provided by the GDP-dependent isoenzyme of succinate thiokinase. This ensures that oxaloacetate will not be withdrawn from the cycle for gluconeogenesis if this would lead to depletion of citric acid cycle intermediates, and hence reduced generation of ATP.
Net transfer into the cycle occurs as a result of several reactions. Among the most important of such anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase (Figure 16–4). This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts as both an allosteric activator of pyruvate carboxylase and an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate. Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate. Glutamate and glutamine are important anaplerotic substrates because they yield α-ketoglutarate as a result of the reactions catalyzed by glutaminase and glutamate dehydrogenase. Transamination of aspartate leads directly to the formation of oxaloacetate, and a variety of compounds that are metabolized to yield propionyl CoA, which can be carboxylated and isomerized to succinyl CoA are also important anaplerotic substrates.
Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and α-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate; arginine, histidine, glutamine, and proline yield α-ketoglutarate; isoleucine, methionine, and valine yield succinyl-CoA; tyrosine and phenylalanine yield fumarate (see Figure 16–4).
The citric acid cycle itself does not provide a pathway for the complete oxidation of the carbon skeletons of amino acids that give rise to intermediates such as α-ketoglutarate, succinyl CoA, fumarate and oxaloacetate, because this results in an increase in the amount of oxaloacetate. For complete oxidation to occur, oxaloacetate must undergo phosphorylation and carboxylation to phosphoenolpyruvate (at the expense of GTP) then dephosphorylation to pyruvate (catalyzed by pyruvate kinase) and oxidative decarboxylation to acetyl Co (catalyzed by pyruvate dehydrogenase).
In ruminants, whose main metabolic fuel is short-chain fatty acids formed by bacterial fermentation, the conversion of propionate, the major glucogenic product of rumen fermentation, to succinyl-CoA via the methylmalonyl-CoA pathway (see Figure 19–2) is especially important.
The Citric Acid Cycle Takes Part in Fatty Acid Synthesis
Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis in nonruminants (Figure 16–5). (In ruminants, acetyl-CoA is derived directly from acetate.) Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosolic pathway; the mitochondrial membrane is impermeable to acetyl-CoA. For acetyl-CoA to be available in the cytosol, citrate is transported from the mitochondrion to the cytosol, then cleaved in a reaction catalyzed by citrate lyase (Figure 16–5). Citrate is only available for transport out of the mitochondrion when aconitase is inhibted by its product and therefore saturated with its substrate, so that citrate cannot be channeled directly from citrate synthase onto aconitase. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle.
Participation of the citric acid cycle in provision of cytosolic acetyl CoA for fatty acid synthesis from glucose. See also Figure 23–5.
The oxaloacetate released by citrate lyase cannot reenter the mitochondrion, but is reduced to malate, at the expense of NADH, and the malate undergoes oxidative decarboxylation to pyruvate, reducing NADP+ to NADPH. This reaction, catalyzed by the malic enzyme, is the source of half the NADPH required for fatty acid synthesis (the remainder is provided by the pentose phosphate pathway, Chapter 20). Pyruvate enters the mitochondrion and is carboxylated to oxaloacetate by pyruvate carboxylase, an ATP-dependent reaction in which the coenzyme is the vitamin biotin.
Regulation of the Citric Acid Cycle Depends Primarily on a Supply of Oxidized Cofactors
In most tissues, where the primary role of the citric acid cycle is in energy-yielding metabolism, respiratory control via the respiratory chain and oxidative phosphorylation regulates citric acid cycle activity (see Chapter 13). Thus, activity is immediately dependent on the supply of NAD+, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the rate of utilization of ATP in chemical and physical work. In addition, individual enzymes of the cycle are regulated. The main sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. The dehydrogenases are activated by Ca2+, which increases in concentration during contraction of muscle and during secretion by other tissues, when there is increased energy demand. In a tissue such as brain, which is largely dependent on carbohydrate to supply acetyl-CoA, control of the citric acid cycle may occur at pyruvate dehydrogenase. Several enzymes are responsive to the energy status as shown by the [ATP]/[ADP] and [NADH]/[NAD+] ratios. Thus, there is allosteric inhibition of citrate synthase by ATP and long-chain fatty acyl-CoA. Allosteric activation of mitochondrial NAD-dependent isocitrate dehydrogenase by ADP is counteracted by ATP and NADH. The α-ketoglutarate dehydrogenase complex is regulated in the same way as is pyruvate dehydrogenase (Figure 17–6). Succinate dehydrogenase is inhibited by oxaloacetate, and the availability of oxaloacetate, as controlled by malate dehydrogenase, depends on the [NADH]/[NAD+] ratio. Since the Km of citrate synthase for oxaloacetate is of the same order of magnitude as the intramitochondrial concentration, it is likely that the concentration of oxaloacetate controls the rate of citrate formation.
Hyperammonemia, as occurs in advanced liver disease and a number of (rare) genetic diseases of amino acid metabolism, leads to loss of consciousness, coma and convulsions, and may be fatal. This is largely because of the withdrawal of α-ketoglutarate to form glutamate (catalyzed by glutamate dehydrogenase) and then glutamine (catalyzed by glutamine synthetase), leading to lowered concentrations of all citric acid cycle intermediates, and hence reduced generation of ATP. The equilibrium of glutamate dehydrogenase is finely poised, and the direction of reaction depends on the ratio of NAD+: NADH and the concentration of ammonium ions. In addition, ammonia inhibits α-ketoglutarate dehydrogenase, and possibly also pyruvate dehydrogenase.