LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

16. THE CITRIC ACID CYCLE

16.3. Regulation of the Citric Acid Cycle

As we saw in Chapter 15, The regulation of Key Enzymes in metabolic pathways—through both allosteric modulators and covalent modification—ensures that intermediates are produced at a rate matching The Cell's steady-state needs, avoiding wasteful overproduction. The flux of carbon atoms from Pyruvate into and through The Citric Acid Cycle is tightly controlled at two main junctions: The conversion of pyruvate to the cycle's starting metabolite, acetyl-CoA (catalyzed by the pyruvate dehydrogenase complex), and The entry of acetyl-CoA into the cycle (the citrate synthase reaction). Because acetyl-CoA is generated not only by the PDH complex but also via the Oxidation of Fatty acids and Certain Amino Acids in most Cells, the availability of these pathway intermediates plays a crucial role in regulating both pyruvate oxidation and The Citric Acid cycle. In addition, the cycle is regulated at the steps catalyzed by isocitrate dehydrogenase and $\alpha$-ketoglutarate dehydrogenase.

The Formation of Acetyl-CoA by the Pyruvate Dehydrogenase Complex Is Regulated by Allosteric and Covalent Interactions

In mammals, the PDH complex is strongly inhibited by ATP, as well as by acetyl-CoA and NADH—the direct products of the complex-catalyzed reaction (Fig. 16-18). This allosteric inhibition of pyruvate oxidation is significantly amplified in the presence of long-chain Fatty acids. Conversely, AMP, CoA, and NAD⁺, which accumulate when the flux of acetate into the citric acid cycle is very low, act as allosteric activators of the PDH complex. Thus, this enzymatic activity decreases when fuel molecules (such as Fatty Acids and acetyl-CoA) are abundant and when the cellular ratios of [ATP]/[ADP] and [NADH]/[NAD⁺] are high, and it increases again when cellular energy demands rise and a greater influx of acetyl-CoA into the citric acid cycle is required.

Class="center">Fig. 16-18. Regulation of metabolite flux from the PDH complex into the citric acid cycle in mammals. Allosteric inhibition of the PDH complex occurs at high [ATP]/[ADP], [NADH]/[NAD⁺], and [acetyl-CoA]/[CoA] ratios, signaling an ample cellular energy reserve. When these ratios decrease, pyruvate oxidation undergoes allosteric activation. The rate of metabolite flux through the citric acid cycle can be limited by the availability of citrate synthase substrates (oxaloacetate and acetyl-CoA) or NAD⁺, which is consumed during its conversion to NADH, thereby slowing the three NAD-dependent oxidative steps. Feedback inhibition by succinyl-CoA, citrate, and ATP also dampens the cycle by throttling its early stages. In Muscle tissue, Ca²⁺ ions serve as a signal for Muscle contraction, stimulating energy production to replenish the ATP consumed during contraction, as shown in the figure.

In mammals, these allosteric regulatory mechanisms are complemented by a second tier of control involving covalent protein modification. The PDH complex is inhibited via the reversible phosphorylation of a specific Ser residue on one of the two subunits of the E₁ protein. As noted earlier, the mammalian PDH complex contains not only the E₁, E₂, and E₃ Proteins, but also two regulatory proteins whose sole function is to govern complex activity. A specific protein kinase phosphorylates and thereby inactivates E₁, while a specific phosphoprotein phosphatase removes the phosphoryl group, thus reactivating E₁. The kinase is activated through allosteric interaction with ATP: when ATP concentrations are high (indicating adequate energy stores), the PDH complex is inactivated via the phosphorylation of E₁. As ATP levels drop, kinase activity declines, and the phosphatase strips the phosphoryl group from E₁, activating the complex.

In plants, the PDH complex located in the mitochondrial matrix and Plastids is inhibited by the products of its catalyzed reaction—NADH and acetyl-CoA. Furthermore, plant enzymatic activity is also regulated by reversible phosphorylation: pyruvate inhibits the kinase, thereby activating the PDH complex, whereas NH₄⁺ stimulates the kinase, promoting complex inactivation. In E. coli cells, the PDH complex is regulated by allosteric interactions in much the same way as in mammalian cells, but regulation via phosphorylation appears to be absent.

The Citric Acid Cycle Is Regulated at Three Exergonic Steps

Metabolite flux through the citric acid cycle is rigorously regulated. The overall pathway rate is determined by three factors: substrate availability, product inhibition, and allosteric feedback inhibition of the enzymes catalyzing the Cytology/cytology/16.html">Early stages of the cycle.

Under certain conditions, any of the three exergonic steps of the cycle—catalyzed by citrate synthase, isocitrate dehydrogenase, or $\alpha$-ketoglutarate dehydrogenase (Fig. 16-18)—can become rate-limiting for the entire process. The cellular availability of citrate synthase substrates (acetyl-CoA and oxaloacetate) varies with conditions, which may limit the rate of citrate formation. NADH generated from The oxidation of isocitrate and $\alpha$-ketoglutarate can accumulate under specific conditions; According to the law of mass action, high [NADH]/[NAD⁺] ratios strongly inhibit both dehydrogenase reactions. The malate dehydrogenase reaction in the cell also operates near steady-state conditions (its rate being constrained by substrate concentrations)—thus, at elevated [NADH]/[NAD⁺] ratios, the oxaloacetate concentration remains low, keeping the rate of the cycle's first step sluggish. Product accumulation restrains all three rate-limiting Stages of the cycle: succinyl-CoA inhibits $\alpha$-ketoglutarate dehydrogenase (as well as citrate synthase), citrate blocks citrate synthase, and ATP inhibits both citrate synthase and isocitrate dehydrogenase. The inhibitory effect of ATP on citrate synthase is relieved in the presence of ADP, an allosteric activator of this enzyme. In vertebrate muscle, Ca²⁺ ions—which signal muscle contraction and a heightened need for ATP—activate isocitrate dehydrogenase, $\alpha$-ketoglutarate dehydrogenase, and the PDH complex alike. In short, the concentrations of citric acid cycle substrates and intermediates set the flux through this pathway at a level that ensures optimal concentrations of ATP and NADH.

Under normal conditions, the rates of Glycolysis and the citric acid cycle are coupled such that glucose yields only The amount of pyruvate required to supply the citric acid cycle with its necessary "fuel"—acetyl groups for acetyl-CoA. The concentrations of pyruvate, lactate, and acetyl-CoA are generally maintained at a steady state. The rate of glycolysis is brought into alignment with the rate of the citric acid cycle not only through inhibition by high levels of ATP and NADH (which typically occur during glucose oxidation via glycolysis and Respiration), but also through shifts in citrate concentration. Citrate, the product of the first step of the citric acid cycle, serves as a potent allosteric inhibitor of Phosphofructokinase-1 during glycolysis (see Fig. 15-14).

Substrate Channeling, Characteristic of Multienzyme Complexes, Can Occur in the Citric Acid Cycle

The Enzymes of the citric acid cycle are generally described as soluble Components of the mitochondrial matrix (with the exception of membrane-bound succinate dehydrogenase), yet mounting evidence suggests that these enzymes exist as multienzyme complexes within Mitochondria. The classical enzymological approach—isolating and purifying individual enzymes from disrupted cell extracts—has been successfully used to study the citric acid cycle enzymes. However, Cell Disruption inevitably disrupts complex intracellular architecture: specifically, the noncovalent bonds formed by reversible protein-protein (enzyme-enzyme) interactions within cellular structures such as membranes, microtubules, or microfilaments. When cells are disrupted, their contents, including enzymes, undergo a 100- to 1,000-fold dilution (Fig. 16-19).

Fig. 16-19. Dilution of a solution containing a complex of noncovalently linked proteins (in this case, comprising three enzymes) promotes the dissociation of the complex into its individual components.

A body of evidence indicates that multienzyme complexes within the cell facilitate the efficient handoff of a product from one enzymatic reaction directly to the next enzyme in the metabolic pathway (such complexes have been termed metabolons). Certain citric acid cycle enzymes form supramolecular complexes; they associate with The inner mitochondrial membrane, or their diffusion rate in the mitochondrial matrix is lower than that of individual enzymes in free solution. Compelling evidence exists for substrate channeling within multienzyme complexes in other metabolic pathways, and many enzymes traditionally viewed as "soluble" actually function within The Cell as highly organized complexes that execute intermediate channeling. We will encounter further Examples of channeling in Chapter 22 when we discuss Amino Acid and nucleotide Biosynthesis.

Certain Mutations in Genes Encoding Citric Acid Cycle Enzymes Promote Cancer

Severe Metabolic Disorders affecting the regulation of critical processes like the citric acid cycle can give rise to serious pathologies. Mutations in the genes for citric acid cycle enzymes are exceedingly rare in humans and other mammals, but when they do occur, the consequences are profoundly dangerous. A defect in the fumarase Gene leads to smooth muscle tumors (leiomyomas) and Kidney tumors, whereas mutations in the succinate dehydrogenase gene result in adrenal tumors (pheochromocytomas). In cell cultures harboring these mutations, fumarate (in fumarase deficiency) or succinate (to a lesser extent in succinate dehydrogenase deficiency) accumulates, which in turn activates Hypoxia-inducible METABOLISM/31.html">Transcription factor 1$\alpha$ (HIF-1$\alpha$; see Box 14-1). Tumor development is likely triggered by a state of pseudohypoxia. Cells carrying these mutations exhibit upregulated expression of genes normally regulated by HIF-1$\alpha$. This pathogenic role of fumarase and succinate dehydrogenase Gene Mutations indicates that these genes function as Tumor Suppressors (see p. 670, Vol. 1). ■

Summary of Section 16.3. Regulation of the Citric Acid Cycle

■ The overall rate of the citric acid cycle is governed by the rate of pyruvate conversion to acetyl-CoA, as well as by the flux of metabolites through the steps catalyzed by citrate synthase, isocitrate dehydrogenase, and $\alpha$-ketoglutarate dehydrogenase. In turn, these fluxes depend heavily on substrate and product concentrations: the end products ATP and NADH inhibit the reactions, whereas the substrates NAD⁺ and ADP stimulate them.

■ The formation of acetyl-CoA—the initial substrate of the citric acid cycle—by the PDH complex is subject to allosteric inhibition by metabolites whose excess signals an abundant level of metabolic energy in the cell (ATP, acetyl-CoA, NADH, and fatty acids), and is stimulated by metabolites whose accumulation signals an energy deficit (AMP, NAD⁺, CoA).

■ In enzyme complexes acting sequentially in a specific metabolic pathway, channeling of metabolic intermediates is possible.