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

PART II. BIOENERGETICS AND METABOLISM

18. AMINO ACID OXIDATION AND THE PRODUCTION OF UREA

18.2. Nitrogen Excretion and the Urea Cycle

When amino groups (Fig. 18-10) are not required for the synthesis of new Amino Acids or other nitrogen-containing products, they are converted into a final excretory product. Most aquatic organisms, such as bony fish, are ammonotelic, meaning they excrete amino nitrogen as ammonia. Toxic ammonia is simply released into the surrounding Water. Terrestrial animals, however, require pathways that minimize toxicity and water loss. Terrestrial animals are predominantly ureotelic, excreting amino nitrogen as urea; birds and reptiles are uricotelic, excreting amino nitrogen as uric acid. (The uric acid biosynthetic pathway is shown in Fig. 22-45.) Plants utilize virtually all of their amino groups and excrete nitrogen only in very rare instances.

In ureotelic organisms, ammonia entering the hepatocyte Cell/35.html">Mitochondria is converted into urea via the Urea Cycle. This pathway was discovered in 1932 by Hans Krebs (who later also discovered The Tricarboxylic Acid Cycle) and his medical student colleague Kurt Henseleit. Urea production occurs almost exclusively in the Liver, where the bulk of metabolic ammonia ultimately converges. Urea enters the bloodstream, travels to the Kidneys, and is excreted in the urine. We will examine The process of Urea formation in this section.

Urea is formed from ammonia in five enzymatic reactions

The urea cycle begins in the liver mitochondria, but the three subsequent steps take place in the Cytosol; the cycle thus spans two cellular compartments (Fig. 18-10). The first amino group to enter the urea cycle is generated in the mitochondrial matrix from ammonium derived from the pathways described earlier. The liver may also receive some ammonium from the intestine via the portal vein, resulting from Bacterial Amino Acid oxidation. Whatever the source, NH⁺₄ generated in the liver mitochondria promptly combines with CO₂ (as HCO^-₃), produced by mitochondrial Respiration, to form carbamoyl phosphate in the matrix (Fig. 18-11a; see also Fig. 18-10). This ATP-dependent reaction is catalyzed by the regulatory enzyme carbamoyl phosphate synthetase I (described below). The mitochondrial form of this enzyme is distinct from the cytosolic form (II), which serves a specialized function in pyrimidine Biosynthesis (Chapter 22).

Carbamoyl phosphate, functioning as an activated carbamoyl group donor, now enters the urea cycle. This cycle consists of four enzymatic steps. First, carbamoyl phosphate transfers its carbamoyl group to Ornithine, yielding citrulline and releasing P_i (Fig. 18-10, step (1)). The Role of ornithine is analogous to that of oxaloacetate in the tricarboxylic acid cycle, acting as a carrier that accepts incoming groups with each turn of the cycle. The reaction is catalyzed by ornithine transcarbamoylase, and the resulting citrulline leaves the mitochondria for the cytosol.

Figure 18-10. The urea cycle and the reactions that precede The entry of amino groups into the cycle.

Class="center">The Enzymes catalyzing these reactions (whose names are given in the text) are distributed between the mitochondrial matrix and the cytosol. One amino group enters the urea cycle as carbamoyl phosphate, formed in the matrix; the other enters as aspartate, synthesized in the matrix via the Transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. The urea cycle consists of four stages: (1) Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group into the cycle); citrulline is then transported into the cytosol. (2) Formation of argininosuccinate via the citrullyl-AMP intermediate (entry of the second amino group into the cycle). (3) Formation of Arginine from argininosuccinate, releasing fumarate, which feeds into The Citric Acid Cycle. (4) Formation of urea, which simultaneously regenerates ornithine. The pathways by which substrates enter the hepatocyte mitochondrial matrix are discussed in Section 18.1.

The second amino group is now contributed by aspartate (which was generated in the mitochondria via transamination and transported into the cytosol). Through a Condensation reaction between the amino group of aspartate and the ureido group of citrulline, argininosuccinate is formed (step (2) in Fig. 18-10). This cytosolic reaction, catalyzed by argininosuccinate synthetase, consumes ATP and proceeds via a citrullyl-AMP intermediate (Fig. 18-11c). Argininosuccinate is subsequently cleaved by argininosuccinase (step (3) in Fig. 18-10) to yield free arginine and fumarate; the latter is directed into the mitochondria to replenish the pool of tricarboxylic acid cycle intermediates. This is the only reversible step in the urea cycle. In the final reaction of the cycle (step (4)), the cytosolic enzyme arginase cleaves arginine to yield urea and ornithine. Ornithine is then transported back into the mitochondria to initiate the next turn of the cycle.

Figure 18-11. Reaction mechanism. Nitrogen-incorporating reactions in urea synthesis. The nitrogen atoms of urea are acquired in two separate reactions, each requiring ATP. (a) In the reaction catalyzed by carbamoyl phosphate synthetase I, the first nitrogen atom comes from ammonium. The terminal phosphate groups of two ATP molecules are consumed to form a single molecule of carbamoyl phosphate; in other words, this reaction involves two activation steps, (1) and (3). Mechanism of carbamoyl phosphate synthetase I. (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen atom is contributed by aspartate. The oxygen of the citrulline ureido group is activated by the attachment of AMP in step (1), which facilitates the subsequent binding of aspartate in step (2). Mechanism of argininosuccinate synthetase

As noted in Chapter 16, the enzymes of many Metabolic pathways are channeled (p. 187) such that the product of one enzymatic reaction is passed directly to the next enzyme in the pathway. In the urea cycle, mitochondrial and cytosolic enzymes are channeled in this very manner. Citrulline exiting the mitochondria does not mix into the general pool of cytosolic metabolites, but is delivered directly to the Active Site of argininosuccinate synthetase. This substrate channeling continues for argininosuccinate, arginine, and ornithine. Only urea is released into the general cytosolic metabolite pool.

The Citric Acid cycle and the urea cycle can be interconnected

Because the fumarate produced in the argininosuccinase reaction is also an intermediate of the citric acid cycle, these two cycles are linked into a larger network often referred to as the "Krebs bicycle" (Fig. 18-12). However, each cycle can operate autonomously, and the cross-talk between them depends on The transport of Key Intermediates across the mitochondrial membrane. Several citric acid cycle enzymes, including fumarase (fumarate hydratase) and malate dehydrogenase (p. 199), exist as distinct isozymes localized in the cytosol. Fumarate generated during cytosolic arginine synthesis can thus be converted to malate in the cytosol, and these intermediates can either undergo further cytosolic METABOLISM or be transported into the mitochondria for use in the citric acid cycle. Conversely, aspartate produced in the mitochondria via transamination between oxaloacetate and glutamate can be transported to the cytosol, where it serves as a nitrogen donor in the urea cycle reaction catalyzed by argininosuccinate synthetase. These reactions, which constitute the aspartate-argininosuccinate shunt, provide crucial metabolic bridges connecting the PATHWAYS OF AMINO acid degradation and amino group disposal.

Figure 18-12. Interconnection between the urea cycle and the citric acid cycle. These intersecting cycles have been termed the "Krebs bicycle." The pathway linking the citric acid cycle to the urea cycle is known as the aspartate-argininosuccinate shunt; it integrates the pathways for the degradation of amino groups and carbon skeletons. The links between the cycles are even more intimate than indicated by the arrows. For example, certain citric acid cycle enzymes, such as fumarase and malate dehydrogenase, possess both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol—whether via the urea cycle, Purine Biosynthesis, or other processes—can be converted to malate, which is either utilized in the cytosol or transported into the mitochondria (via the malate-aspartate shuttle; see Fig. 19-29) to participate in the citric acid cycle.

Enzymatic activities in the urea cycle are regulated at two levels

In animals, the flux of nitrogen through the urea cycle varies depending on dietary intake. When the diet is rich in Proteins, the carbon skeletons of Amino acids are catabolized for energy, yielding large amounts of urea from the surplus amino groups. Urea production is also markedly increased during prolonged starvation, when The breakdown of Muscle protein is accelerated to sustain the Organism's metabolic energy needs.

To accommodate shifting demands for urea cycle activity, the rates of Synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver undergo long-term adaptation. In starving animals and those maintained on high-protein diets, all five enzymes are expressed at significantly higher levels than in well-fed animals subsisting primarily on CARBOHYDRATES and fats. Conversely, animals on low-protein diets exhibit suppressed synthesis of urea cycle enzymes. These adjustments in enzyme expression levels provide slow, long-term Regulation of Urea cycle capacity.

Rapid Allosteric Regulation of at least one key enzyme modulates flux through the urea cycle over a short time scale. The first enzyme of the pathway, carbamoyl phosphate synthetase I, is allosterically activated by N-acetylglutamate, which is synthesized from acetyl-CoA and glutamate by the action of N-acetylglutamate synthetase (Fig. 18-13). In plants and microorganisms, this enzyme catalyzes the initial step in the de novo biosynthesis of arginine from glutamate (see Fig. 22-10), but in mammals, N-acetylglutamate synthetase activity serves a purely regulatory function (mammals lack the requisite downstream enzymes to convert glutamate to arginine). The intracellular level of N-acetylglutamate is determined by the concentrations of glutamate and acetyl-CoA (substrates for N-acetylglutamate synthetase) and arginine (an activator of N-acetylglutamate synthase and, consequently, of the urea cycle).

Figure 18-13. Synthesis of N-acetylglutamate and its activation of carbamoyl phosphate synthetase I.

Metabolic pathway intersections reduce The Energetic Cost of urea synthesis

If we consider the urea cycle in isolation, we find that the synthesis of a single molecule of urea consumes four high-energy phosphate groups (Fig. 18-10). Two molecules of ATP are required to form carbamoyl phosphate, and one ATP is required for argininosuccinate, with the latter ATP being hydrolyzed to AMP and PP_i, which then dissociates to yield two P_i. The overall equation for the urea cycle is:

2NH₄⁺+ НСO₃^- + 3АТР⁴- +Н₂O —> мочевина + 2ADP^3-+ 4P_i^2- + АМР^2- + 2Н⁺

However, the urea cycle also involves the sequential conversion of oxaloacetate to fumarate (via aspartate), and during the regeneration of oxaloacetate (Fig. 18-12), NADH is produced in the malate dehydrogenase reaction. During mitochondrial respiration, each molecule of NADH can yield up to 2.5 molecules of ATP (Chapter 19), which significantly lowers the net energetic cost of urea synthesis.

Genetic Defects in the urea cycle can be life-threatening

Individuals with genetic defects in any of the enzymes involved in urea formation cannot tolerate a protein-rich diet. Any excess dietary amino acids beyond the body's minimum daily requirements for Protein Synthesis undergo deamination in the liver to yield free ammonia. Because this ammonia cannot be converted into urea, it enters the bloodstream and, as we have noted, is highly toxic. The absence of even a single urea cycle enzyme can lead to hyperammonemia or the accumulation of a specific cycle intermediate, depending on which enzyme is non-functional. Knowing that most urea cycle reactions are irreversible, the defective enzyme can be identified by determining which intermediate is present in abnormally high concentrations in the Blood and/or urine. Although Amino Acid Breakdown has severe consequences for individuals with urea cycle defects, simply abstaining from dietary protein is not a proper Treatment. Humans are unable to synthesize nearly half of the 20 standard amino acids, and these Essential Amino Acids (Table 18-1) must be obtained from the diet.

Table 18-1. Nonessential and essential amino acids in humans and the albino rat

* Required in small amounts during infancy, in growing animals, and/or occasionally during illness.

Various therapeutic strategies are available for managing urea cycle defects. The careful dietary administration of aromatic acids—such as benzoate or phenylbutyrate—can reduce blood ammonia levels. Benzoate is converted to benzoyl-CoA, which then combines with glycine to form hippurate (Fig. 18-14, left). This reaction consumes glycine, which must be replenished, thereby pulling ammonia into the glycine synthetase pathway. Phenylbutyrate is converted to phenylacetate via β-oxidation. Phenylacetate is subsequently converted to phenylacetyl-CoA, which combines with glutamine to form phenylacetylglutamine (Fig. 18-14, right). Consequently, the removal of glutamine drives its further synthesis by Glutamine Synthetase (see Equation 22-1) in a reaction that consumes ammonia. Both hippurate and phenylacetylglutamine are non-toxic compounds that are excreted in the urine. While the pathways shown in Fig. 18-14 account for only a minor fraction of normal metabolism, they are markedly upregulated upon the ingestion of large amounts of aromatic acids.

Figure 18-14. Treatment of urea cycle enzyme deficiencies. The aromatic acids benzoate and phenylbutyrate are administered orally and conjugate with glycine and glutamine, respectively. The resulting products are excreted in the urine. The subsequent de novo synthesis of glycine and glutamine to replenish the pools of these intermediates removes ammonium from the blood.

Other treatments are more specific to particular enzyme deficiencies. A deficiency in N-acetylglutamate synthase leads to a lack of the natural activator for carbamoyl phosphate synthetase I (Fig. 18-13). This condition can be bypassed by administering carbamoyl glutamate, an analog of N-acetylglutamate that effectively activates carbamoyl phosphate synthetase I.

A high-arginine diet is beneficial in treating deficiencies of ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinase. Most of these therapies must be accompanied by strict dietary management and essential amino acid supplementation. In rare cases of arginase deficiency, the enzyme's substrate, arginine, must be rigorously excluded from the diet. ■

Summary of Section 18.2 Nitrogen Excretion and the Urea Cycle

■ Ammonia is highly toxic to animal Tissues. In the urea cycle, ornithine combines with ammonia (in the form of carbamoyl phosphate) to yield citrulline. A second amino group is transferred to citrulline from aspartate, ultimately producing arginine—the immediate precursor of urea. Arginase catalyzes the Hydrolysis of arginine to urea and ornithine, thus regenerating ornithine with each turn of the cycle.

■ The urea cycle is linked to a sequence of interconversions extending from oxaloacetate to fumarate, both of which are Intermediates of the citric acid cycle. Thus, these two cycles intersect.

■ The catalytic activities of the urea cycle are regulated at two levels: through the control of enzyme synthesis and via allosteric Regulation of the enzyme that catalyzes carbamoyl phosphate formation.