Principles of Biochemistry Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
The Citric Acid Cycle
Intermediates of the citric acid cycle are also utilized in other metabolic pathways, and their depletion is continuously replenished.

The Citric Acid Cycle is one of the amphibolic pathways (Section 13.7). It is used not only for oxidative Catabolism—i.e., The breakdown of CARBOHYDRATES, Fatty acids, and Amino Acids—but can also serve as the initial stage for many biosynthetic pathways, for which it provides crucial precursors. Through the action of several key auxiliary Enzymes, certain intermediates of The Citric Acid cycle, primarily a-ketoglutarate, succinate, and oxaloacetate, can be withdrawn from the cycle and utilized as amino acid precursors (Chapter 22). At first glance, this might suggest that The rate of the citric acid cycle should decline, since such an efflux of intermediates would lower their intracellular concentrations. In reality, however, this does not happen, because the depletion of cycle intermediates is continuously replenished by another set of enzymes. Under normal conditions, the reactions that draw intermediates out of the cycle and those that replenish them exist in a dynamic equilibrium, so that the concentrations of these intermediates in the Cell/35.html">Mitochondria remain relatively constant.

Special enzymatic reactions that replenish the pool of citric acid cycle intermediates are known as anaplerotic (“filling-up”) reactions. The most important reaction of this type in animal Tissues is the enzymatic carboxylation of Pyruvate by CO₂ to form oxaloacetate (Fig. 16-16); this reversible reaction is catalyzed by the enzyme pyruvate carboxylase

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If the supply of oxaloacetate or any other cycle intermediate becomes insufficient for the citric acid cycle, pyruvate carboxylation is stimulated, leading to an increased pool of oxaloacetate. The enzymatic attachment of a carboxyl group to a pyruvate molecule requires energy, which is provided by the coupled Cleavage of ATP to ADP and inorganic phosphate. Because the overall reaction is accompanied by only a negligible change in Standard Free energy, we can conclude that the free energy required to attach the carboxyl group to pyruvate is approximately equal to the free energy released during ATP Hydrolysis.

Fig. 16-16. The pyruvate carboxylase reaction and its stimulation by the positive modulator acetyl-CoA. The incorporated CO₂ is highlighted on a red Background.

Pyruvate carboxylase is a highly complex enzyme with a Molecular Weight of approximately 650,000. The enzyme molecule contains four prosthetic groups, each consisting of a molecule of the vitamin biotin (Section 10.9) covalently linked (via a peptide bond) to the e-amino group of a specific Lysine residue located at the Active Site (Fig. 16-17). Free CO₂, the precursor of the new oxaloacetate carboxyl group, is first activated by attaching to one of the nitrogen atoms in the biotin molecule. This activation, which requires the consumption of ATP, constitutes The First stage of the reaction catalyzed by pyruvate carboxylase (where E denotes the enzyme):

In the second stage, which also takes place at the Active Site of the enzyme, the new carboxyl group—covalently bound to the enzyme's prosthetic group—is transferred to pyruvate to yield oxaloacetate (Fig. 16-16):

Fig. 16-17. The prosthetic group of pyruvate carboxylase. The carboxyl group of biotin forms a peptide bond with the e-amino group of a lysine residue within the active site of the enzyme. CO₂ is activated, forming an N-carboxy derivative of the biotinyl prosthetic group. This carboxyl group—acting as the direct CO₂ donor for pyruvate—is then transferred to pyruvate.

Pyruvate carboxylase belongs to the class of regulatory enzymes. In the absence of acetyl-CoA, which acts as a positive modulator, the rate of the forward reaction leading to oxaloacetate formation is very low (Fig. 16-16). Conversely, an excess of acetyl-CoA—which supplies the “fuel” for the citric acid cycle—stimulates the pyruvate carboxylase reaction, resulting in increased oxaloacetate production and enabling the cycle to process more acetyl-CoA via the citrate synthase reaction.

The pyruvate carboxylase reaction is the primary anaplerotic reaction in The Liver and Kidneys. In the myocardium and skeletal Muscles, different anaplerotic reactions take place. One such reaction is catalyzed by phosphoenolpyruvate carboxykinase (Chapter 20)

This reaction involves the cleavage of phosphoenolpyruvate—a high-energy phosphorylated compound generated during Glycolysis. The released energy is utilized for carboxylation to form oxaloacetate, while the remainder is conserved in the form of GTP.