Amino Acid Oxidation and the Production of Urea

CHAPTER 18 AMINO ACID OXIDATION AND THE PRODUCTION OF UREA CO2 and H2O in photosynthesis is generally their sole energy source. Amino acid concentrations in plant tissues are carefully regulated to just meet the requirements for biosynthesis of proteins, nucleic acids, and other molecules needed to support growth. Amino acid catabolism does occur in plants, but only to produce metabolites for other biosynthetic pathways. Amino acid oxidation pathways can seem complex, and they are best understood in the context of five principles: The many paths for amino acid catabolism have two broad parts, one involving the amino groups and the other involving the carbon skeletons. All of the pathways for amino acid degradation include a key step, always involving a pyridoxal phosphate cofactor, in which the α -amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism. The carbon skeletons are broken down to citric acid cycle intermediates (Fig. 18-1). Four amino acids — alanine, glutamate, glutamine, and aspartate — play key roles in the transport and distribution of amino groups. All are present in relatively high concentrations in one or many mammalian tissues. All are readily converted to key citric acid cycle intermediates. Metabolic pathways are not distinct. The various pathways for amino acid catabolism are elaborately intertwined with other catabolic and anabolic pathways. Free ammonia is toxic. Excess amino groups must be safely excreted. In mammals, the urea cycle serves this purpose. Each amino acid has a different catabolic fate. The varied carbon skeletons of amino acids are broken down via equally varied pathways. All can be oxidized to generate ATP. All but leucine and lysine can contribute to gluconeogenesis when needed. FIGURE 18-1 Overview of amino acid catabolism in mammals. The amino groups and the carbon skeleton take separate but interconnected pathways. In mammals, blood glucose must be supplemented by gluconeogenesis, oen just a few hours aer a meal. Amino acids, particularly alanine and glutamine, can make a significant contribution to the fuel for gluconeogenesis. Amino acids undergo oxidative degradation in three different metabolic circumstances: 1. Amino acids released during normal protein turnover are not needed for new protein synthesis. 2. Ingested amino acids exceed the body’s needs for protein synthesis. 3. Cellular proteins are used as fuel because carbohydrates are either unavailable or not properly utilized due to starvation or uncontrolled diabetes mellitus. The pathways of amino acid catabolism are quite similar in most organisms. The focus of this chapter is on the pathways in vertebrates, because these have received the most research attention. As in carbohydrate and fatty acid catabolism, the processes of amino acid degradation converge on the central catabolic pathways, with the carbon skeletons of most amino acids finding their way to the citric acid cycle. In some cases, the reaction pathways of amino acid breakdown closely parallel steps in the catabolism of fatty acids (see Fig. 17-9). We begin with amino group metabolism and nitrogen excretion, before turning to the fate of the carbon skeletons derived from the amino acids; along the way we see how the pathways are interconnected. 18.1 Metabolic Fates of Amino Groups Nitrogen, N2, is abundant in the atmosphere but is too unreactive for use in most biochemical processes. Reduced nitrogen is essential for life but bioenergetically expensive. Only a few microorganisms can convert N2 to biologically useful forms such as NH3 (Chapter 22). Amino groups are thus used efficiently in biological systems, and the lack of reactive nitrogen can limit growth. If not reused for the synthesis of new amino acids or other nitrogenous products, amino groups are channeled into a single excretory end product (Fig. 18-2). Most aquatic species, such as the bony fishes, are ammonotelic, excreting amino nitrogen as ammonia. The toxic ammonia is simply diluted in the surrounding water. Terrestrial animals require pathways for nitrogen excretion that minimize toxicity and water loss. Most terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea; birds and reptiles are uricotelic, excreting amino nitrogen as uric acid. (The pathway of uric acid synthesis is described in Fig. 22-48.) Plants recycle virtually all amino groups — they excrete nitrogen only under rare circumstances. Reactive nitrogen excreted in any form is rapidly assimilated into the global nitrogen web (see Fig. 22-1), metabolized by microorganisms that are ubiquitous in aqueous and soil environments. FIGURE 18-2 Amino group catabolism. (a) Overview of catabolism of amino groups (shaded) in vertebrate liver. (b) Excretory forms of nitrogen. Excess NH+ 4 is excreted as ammonia, urea, or uric acid. Notice that the carbon atoms of urea and uric acid are highly oxidized; the organism discards carbon only a er extracting most of its available energy of oxidation. Figure 18-2a provides an overview of the catabolic pathways of ammonia and amino groups in vertebrates. Glutamine, glutamate, and alanine are prominent in this scheme. Amino acids derived from dietary protein are the source of most amino groups. Most amino acids are metabolized in the liver. Some of the ammonia generated in this process is recycled and used in biosynthetic pathways where glutamine, glutamate, and aspartate play major roles (Chapter 22). Excess amino groups are either excreted directly or converted to urea or uric acid for excretion, depending on the organism (Fig. 18-2b). In mammals, including marsupials, most excess ammonia generated in other (extrahepatic) tissues travels to the liver for conversion to urea. The special place of glutamate, glutamine, alanine, and aspartate in nitrogen metabolism is not an evolutionary accident. These particular amino acids are the ones most easily converted into citric acid cycle intermediates: glutamate and glutamine to α -ketoglutarate, alanine to pyruvate, and aspartate to oxaloacetate. Glutamate and glutamine are especially important, acting as general collection points for amino groups. In the cytosol of liver cells (hepatocytes), amino groups from most amino acids are transferred to α -ketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH+ 4. Excess ammonia generated in most other tissues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria. Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues. In skeletal muscle, excess amino groups are generally transferred to pyruvate to form alanine, another important molecule in the transport of amino groups to the liver. We begin with a discussion of the breakdown of dietary proteins, then proceed to a general description of the metabolic fates of amino groups. Dietary Protein Is Enzymatically Degraded to Amino Acids In humans, the degradation of ingested proteins to their constituent amino acids occurs in the gastrointestinal tract. Entry of dietary protein into the stomach stimulates the gastric mucosa to secrete the hormone gastrin, which in turn stimulates the secretion of hydrochloric acid by the parietal cells and pepsinogen by the chief cells of the gastric glands (Fig. 18-3a). The acidic gastric juice (pH 1.0 to 2.5) is both an antiseptic, killing most bacteria and other foreign cells, and a denaturing agent, unfolding globular proteins and rendering their internal peptide bonds more accessible to enzymatic hydrolysis. Pepsinogen (Mr40,554), an inactive precursor, or zymogen (p. 220), is converted to active pepsin (Mr34,614) by an autocatalytic cleavage (a cleavage mediated by the pepsinogen itself) that occurs only at low pH. In the stomach, pepsin cleaves long polypeptide chains into a mixture of smaller peptides.

FIGURE 18-3 Part of the human digestive (gastrointestinal) tract. (a) The parietal cells and chief cells of the gastric glands secrete their products in response to the hormone gastrin. Pepsin begins the process of protein degradation in the stomach. (b) The cytoplasm of exocrine cells of the pancreas is completely filled with rough endoplasmic reticulum, the site of synthesis of the zymogens of many digestive enzymes. The zymogens are concentrated in membrane-enclosed transport particles called zymogen granules. When an exocrine cell is stimulated, its plasma membrane fuses with the zymogen granule membrane and zymogens are released into the lumen of the collecting duct by exocytosis. The collecting ducts ultimately lead to the pancreatic duct and from there to the small intestine. (c) In the small intestine, amino acids are absorbed through the epithelial cell layer (intestinal mucosa) of the villi and enter the capillaries. As the acidic stomach contents pass into the small intestine, the low pH triggers secretion of the hormone secretin into the blood. Secretin stimulates the pancreas to secrete bicarbonate into the small intestine to neutralize the gastric HCl, abruptly increasing the pH to about 7. Arrival of peptides in the upper part of the intestine (duodenum) causes release into the blood of the hormone cholecystokinin, which stimulates secretion of several pancreatic proteases with activity optima at pH 7 to 8. Trypsinogen, chymotrypsinogen, and procarboxypeptidases A and B—the zymogens of trypsin, chymotrypsin, and carboxypeptidases A and B—are synthesized and secreted by the exocrine cells of the pancreas (Fig. 18-3b). Trypsinogen is converted to its active form, trypsin, by enteropeptidase, a proteolytic enzyme secreted by intestinal cells. Free trypsin then catalyzes the conversion of additional trypsinogen to trypsin (see Fig. 6-42). Trypsin also activates chymotrypsinogen, the procarboxypeptidases, and proelastase. Why this elaborate mechanism for getting active digestive enzymes into the gastrointestinal tract? Synthesis of the enzymes as inactive precursors protects the exocrine cells from destructive proteolytic attack. The pancreas further protects itself against self-digestion by making a specific inhibitor, a protein called pancreatic trypsin inhibitor (p. 220). Given the key role of trypsin in proteolytic activation pathways, inhibition of trypsin effectively prevents premature production of active proteolytic enzymes within pancreatic cells. Trypsin and chymotrypsin further hydrolyze the peptides that were produced by pepsin in the stomach. This stage of protein digestion is accomplished very efficiently, because pepsin, trypsin, chymotrypsin, and the carboxypeptidases have different catalytic specificities and cleave different sets of peptide bonds (see Table 3-6). The resulting mixture of free amino acids is transported into the epithelial cells lining the small intestine (Fig. 18-3c), through which the amino acids enter the blood capillaries in the villi and travel to the liver. Acute pancreatitis is a disease caused by obstruction of the normal pathway by which pancreatic secretions enter the intestine. The zymogens of the proteolytic enzymes are converted to their catalytically active forms prematurely, inside the pancreatic cells, and attack the pancreatic tissue itself. This causes excruciating pain and damage to the organ that can prove fatal.

Pyridoxal Phosphate Participates in the Transfer of α -Amino Groups to α -Ketoglutarate The first step in the catabolism of most L-amino acids, once they have reached the liver, is removal of the α -amino groups, promoted by enzymes called aminotransferases or transaminases. In these transamination reactions, the α -amino group is transferred to the α -carbon atom of α -ketoglutarate, leaving behind the corresponding α -keto acid analog of the amino acid (Fig. 18-4). There is no net deamination (loss of amino groups) in these reactions, because the α -ketoglutarate becomes aminated as the α -amino acid is deaminated. These are the reactions that effectively collect the amino groups from many different amino acids in the form of L-glutamate. FIGURE 18-4 Enzyme-catalyzed transaminations. In many aminotransferase reactions, α -ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. Although the reaction is shown here in the direction of transfer of the amino group to α -ketoglutarate, it is readily reversible. Cells contain different types of aminotransferases. Many are specific for α -ketoglutarate as the amino group acceptor but differ in their specificity for the L-amino acid. The enzymes are named for the amino group donor (alanine aminotransferase and aspartate aminotransferase, for example). The reactions catalyzed by aminotransferases are freely reversible, having an equilibrium constant of about 1.0 (ΔG′≈ 0kJ/mol). All aminotransferases have pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6, as a prosthetic group. We encountered pyridoxal phosphate in Chapter 15, as a coenzyme in the glycogen phosphorylase reaction, but its role in that reaction is not representative of its usual coenzyme function. Its primary role in cells is in the metabolism of molecules with amino groups. Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of aminotransferases. It undergoes reversible transformations between its aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyridoxamine phosphate, which can donate its amino group to an α -keto acid (Fig. 18-5a). Pyridoxal phosphate is generally covalently bound to the enzyme’s active site through an aldimine (Schiff base) linkage to the ε -amino group of a Lys residue (Fig. 18-5b, c). This linkage is replaced by the amino group of the amino acid as the first step in most PLP-catalyzed reactions (Fig. 18-5d).

FIGURE 18-5 Pyridoxal phosphate, the prosthetic group of aminotransferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyridoxamine phosphate, are the tightly bound coenzymes of aminotransferases. The functional groups are shaded. (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiff-base (aldimine) linkage to a Lys residue at the active site. The steps in the formation of a Schiff base from a primary amine and a carbonyl group are detailed in Figure 14-5. (c, d) Close-up views of the active site of aspartate aminotransferase, with PLP (white, with the phosphoryl group in orange and red). In (c) PLP is in aldimine linkage with the side chain of Lys258 (purple). In (d) PLP is linked to the substrate analog 2- methylaspartate (green) via a Schiff base. [(c, d) Data from PDB ID 1AJS, S. Rhee et al., J. Biol. Chem. 272:17,293, 1997.] Pyridoxal phosphate participates in a variety of reactions in amino acid metabolism (Section 18.3 and Chapter 22), facilitating reactions at the α , β , and γ carbons (C-2 to C-4) of amino acids. Reactions at the α carbon (Fig. 18-6) include racemizations (interconverting L- and D-amino acids) and decarboxylations, as well as transaminations. Pyridoxal phosphate plays the same chemical role in each of these reactions. A bond to the α carbon of the substrate is broken, removing either a proton or a carboxyl group. Pyridoxal phosphate provides resonance stabilization of the otherwise unstable carbanion intermediate (Fig. 18-6, inset). The highly conjugated structure of PLP (an electron sink) permits delocalization of the negative charge. MECHANISM FIGURE 18-6 Some amino acid transformations at the α carbon that are facilitated by pyridoxal phosphate. Pyridoxal phosphate is generally bonded to the enzyme through a Schiff base, also called an internal aldimine. This activated form of PLP readily undergoes transamination to form a new Schiff base (external aldimine) with the α -amino group of the substrate amino acid (see Fig. 18-5b, d). Three alternative fates for the external aldimine are shown: transamination, racemization, and decarboxylation. The PLP–amino acid Schiff base is in conjugation with the pyridine ring, an electron sink that permits delocalization of an electron pair to avoid formation of an unstable carbanion on the α carbon (inset). A quinonoid intermediate is involved in all three types of reactions. The transamination route is especially important in the pathways described in this chapter. This pathway (shown le to right) represents only part of the overall reaction catalyzed by aminotransferases. To complete the process, a second α -keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps (right to le ). Pyridoxal phosphate is also involved in certain reactions at the β and γ carbons of some amino acids (not shown). Aminotransferases (Fig. 18-6a) are classic examples of enzymes catalyzing bimolecular Ping-Pong reactions (see Fig. 6-15b, d), in which the first substrate reacts and the product must leave the active site before the second substrate can bind. Thus, the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an α - keto acid. The incoming α -keto acid then binds, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid. Glutamate Releases Its Amino Group as Ammonia in the Liver As we shall see, the urea cycle begins with free ammonia in the mitochondria of hepatocytes. The delivery of NH+ 4 to these mitochondria is streamlined by collecting the amino groups of many different α -amino acids in the liver in one of two forms: the amino group of L-glutamate or the amide nitrogen of glutamine (Fig. 18-2). As the product of many aminotransferase reactions, glutamate has a central role. In hepatocytes, glutamate is transported from the cytosol into mitochondria. Here, it undergoes oxidative deamination catalyzed by L-glutamate dehydrogenase (Mr330,000) to produce NH+ 4 and α -ketoglutarate. In mammals, this enzyme is present in the mitochondrial matrix. It is unusual in that it can use either NAD+ or NADP+ as the acceptor of reducing equivalents (Fig. 18-7). FIGURE 18-7 Reaction catalyzed by glutamate dehydrogenase. The glutamate dehydrogenase of mammalian liver has the unusual capacity to use either NAD+ or NADP+ as cofactor. The glutamate dehydrogenases of plants and microorganisms are generally specific for one or the other. The mammalian enzyme is allosterically regulated by GTP and ADP. The combined action of an aminotransferase and glutamate dehydrogenase is referred to as transdeamination. A few amino acids bypass the transdeamination pathway and undergo direct oxidative deamination. The fate of the NH+4 produced by any of these deamination processes is discussed in detail in Section 18.2. The α -ketoglutarate formed from glutamate deamination can be used either in the citric acid cycle or for glucose synthesis.

Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen metabolism. Its α - ketoglutarate product can be oxidized as fuel or serve as a glucose precursor in gluconeogenesis. An allosteric enzyme with six identical subunits, its activity is influenced by a complicated array of allosteric modulators. The best-studied of these are the positive modulator ADP and the negative modulator GTP. Although the metabolic rationale for this regulatory pattern has not been elucidated in detail, ADP can signal low glucose levels and GTP is a product of the citric acid cycle that can signal high levels of α - ketoglutarate (see Fig. 16-7). Mutations that alter the allosteric binding site for GTP or otherwise cause permanent activation of glutamate dehydrogenase lead to a human genetic disorder called hyperinsulinism-hyperammonemia syndrome, characterized by hypersecretion of insulin aer a protein meal. This results in elevated levels of ammonia in the bloodstream and hypoglycemia.

Glutamine Transports Ammonia in the Bloodstream Glutamine is the second major source of ammonia in hepatocyte mitochondria and is particularly important for intercellular transport of ammonia. Ammonia is quite toxic to animal tissues (we examine some possible reasons for this toxicity later). Significant amounts are present in blood, but the levels are tightly controlled. In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia. In most animals, much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys. For this transport function, glutamate, critical to intracellular amino group metabolism, is supplanted by L-glutamine. The free ammonia produced in tissues is combined with glutamate to yield glutamine by the action of glutamine synthetase. This reaction requires ATP and occurs in two steps (Fig. 18-8). First, glutamate and ATP react to form ADP and a γ -glutamyl phosphate intermediate, which then reacts with ammonia to produce glutamine and inorganic phosphate. In addition to its transport role, glutamine serves as a source of amino groups in a variety of biosynthetic reactions. Glutamine synthetase is found in all organisms, always playing a central metabolic role. In microorganisms, the enzyme serves as an essential portal for the entry of fixed nitrogen into biological systems. (The roles of glutamine and glutamine synthetase in metabolism are further discussed in Chapter 22.)

FIGURE 18-8 Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. A er transport in the bloodstream, the glutamine enters the liver, and NH+4 is liberated in mitochondria by the enzyme glutaminase. In most terrestrial animals, glutamine in excess of that required for biosynthesis is transported in the blood to the intestine, liver, and kidneys for processing. In these tissues, the amide nitrogen is released as ammonium ion in the mitochondria, where the enzyme glutaminase converts glutamine to glutamate and NH+ 4 (Fig. 18-8). The NH+ 4 from intestine and kidney is transported in the blood to the liver. In the liver, the ammonia from all sources is disposed of by urea synthesis. Some of the glutamate produced in the glutaminase reaction may be further processed in the liver by glutamate dehydrogenase (Fig. 18-7), releasing more ammonia and producing carbon skeletons for metabolic fuel. In metabolic acidosis (p. 621) there is a regulated increase in glutamine processing by the kidneys. Much of the excess NH+ 4 thus produced is not destined for the bloodstream or converted to urea; instead, it is excreted directly into the urine where it forms salts with metabolic acids. The glutamine breakdown thus facilitates removal of those acids in the urine. Bicarbonate produced by the decarboxylation of α -ketoglutarate in the citric acid cycle can also serve as a buffer in blood plasma. Taken together, these effects of glutamine metabolism in the kidney tend to counteract acidosis.

Alanine Transports Ammonia from Skeletal Muscles to the Liver Vigorously contracting skeletal muscles operate anaerobically, producing large amounts of pyruvate and lactate from glycolysis as well as ammonia from protein breakdown. These products must find their way to the liver, where pyruvate and lactate are incorporated into glucose, which is returned to the muscles, and ammonia is converted to urea for excretion. Pyruvate and alanine are readily interconverted via transamination with glutamate by the action of alanine aminotransferase). Thus, alanine largely supplants glutamine in the transport of amino groups from muscle to the liver in a nontoxic form (Fig. 18-2a), ultimately delivering the free ammonia to hepatocyte mitochondria via glutamate in a pathway called the glucose-alanine cycle (Fig. 18- 9). In the cytosol of hepatocytes, alanine aminotransferase acts in reverse to transfer the amino group from alanine to α - ketoglutarate, forming pyruvate and glutamate. Glutamate can then enter mitochondria, where the glutamate dehydrogenase reaction releases NH+4 (Fig. 18-7), or it can undergo transamination with oxaloacetate to form aspartate, another nitrogen donor in urea synthesis, as we shall see. FIGURE 18-9 Glucose-alanine cycle. Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate from skeletal muscle to liver. The ammonia is excreted and the pyruvate is used to produce glucose, which is returned to the muscle. The use of alanine to transport ammonia from skeletal muscles to the liver is another example of the intrinsic economy of living organisms. The energetic burden of gluconeogenesis is imposed on the liver rather than the muscle, and all available ATP in muscle is devoted to muscle contraction. The glucose-alanine cycle, in concert with the Cori cycle (see Box 14-2 and Fig. 23-17), accomplishes this transaction. Ammonia Is Toxic to Animals The catabolic production of ammonia poses a serious biochemical problem, because ammonia is very toxic. The brain is particularly sensitive; damage from ammonia toxicity causes cognitive impairment, ataxia, and epileptic seizures. In extreme cases there is swelling of the brain leading to death. The molecular bases for this toxicity are gradually coming into focus. In the blood, about 98% of ammonia is in the protonated form (NH+4), which does not cross the plasma membrane. The small amount of NH3 present readily crosses all membranes, including the blood-brain barrier, allowing it to enter cells, where much of it becomes protonated and can accumulate inside cells as NH+4. Ridding the cytosol of ammonia requires reductive amination of α -ketoglutarate to glutamate by glutamate dehydrogenase (the reverse of the reaction described earlier; Fig. 18-7) and conversion of glutamate to glutamine by glutamine synthetase. In the brain, only astrocytes — star-shaped cells of the nervous system that provide nutrients, support, and insulation for neurons — express glutamine synthetase. Glutamate and its derivative γ -aminobutyrate (GABA; see Fig. 22-31) are important neurotransmitters; some of the sensitivity of the brain to ammonia may reflect depletion of glutamate in the glutamine synthetase reaction. However, glutamine synthetase activity is insufficient to deal with excess ammonia, or to fully explain its toxicity. Increased [NH+ 4] also alters the capacity of astrocytes to maintain potassium homeostasis across the membrane. NH+ 4 competes with K+ for transport into the cell through the Na+K+ ATPase, resulting in elevated extracellular [K+]. The excess extracellular K+ enters neurons through a symporter, Na+−K+−2Cl− cotransporter 1 (NKCC1), bringing Na+ and 2Cl− with it. Excess Cl− in these neurons alters their response when the neurotransmitter GABA interacts with their GABAA receptors, producing abnormal depolarization and increased neuronal activity that likely account for the neuromuscular incoordination and seizures that oen result from ammonia poisoning. If extracellular [NH+ 4] remains elevated, the perturbation of ion and aquaporin channels in astrocytes causes the cells to swell, resulting in fatal brain edema. As we close this discussion of amino group metabolism, note that we have described several processes that deposit excess ammonia in the mitochondria of hepatocytes (Fig. 18-2). We now look at the fate of that ammonia. SUMMARY 18.1 Metabolic Fates of Amino Groups Humans derive a small fraction of their oxidative energy from the catabolism of amino acids. Proteases degrade ingested proteins in the stomach and small intestine. Most proteases are initially synthesized as inactive zymogens. An early step in the catabolism of amino acids is the separation of the amino group from the carbon skeleton. In most cases, the amino group is transferred to α -ketoglutarate to form glutamate. This transamination reaction requires pyridoxal phosphate, a coenzyme globally involved in amino acid metabolism. Glutamate, a central reservoir of metabolized amino groups, is transported to liver mitochondria, where glutamate dehydrogenase liberates the amino group as ammonium ion (NH+4). Ammonia formed in most other tissues is transported to the liver as the amide nitrogen of glutamine. To make use of the pyruvate and amino groups generated in hardworking skeletal muscle, the pyruvate is converted to alanine and transported to the liver within the glucose-alanine cycle. Free ammonia is toxic. Excess ammonia is oen manifested in serious neurological damage. 18.2 Nitrogen Excretion and the Urea Cycle In ureotelic organisms, the ammonia deposited in the mitochondria of hepatocytes is converted to urea in the urea cycle (Fig. 18-10). This pathway was discovered in 1932 by Hans Krebs (who later also discovered the citric acid cycle) and a medical student associate, Kurt Henseleit. Urea production occurs almost exclusively in the liver and is the fate of most of the ammonia channeled there. The urea passes into the bloodstream and thus to the kidneys and is excreted into the urine. The production of urea now becomes the focus of our discussion. FIGURE 18-10 The urea cycle and reactions that feed amino groups into the cycle. The enzymes catalyzing these reactions (named 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, formed in the matrix by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase. The urea cycle consists of four steps: Formation of citrulline from ornithine and carbamoyl phosphate (entry of the first amino group); the citrulline passes into the cytosol. Formation of argininosuccinate through a citrullyl-AMP intermediate (entry of the second amino group). Formation of arginine from argininosuccinate; this reaction releases fumarate, which enters the citric acid cycle. Formation of urea; this reaction also regenerates ornithine. Urea Is Produced from Ammonia in Five Enzymatic Steps The urea cycle begins inside liver mitochondria, but three of the 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 derived from ammonia in the mitochondrial matrix — most of this NH+ 4 arises by the pathways described in Section 18.1. The liver also receives some ammonia via the portal vein from the intestine, from the bacterial oxidation of amino acids. Whatever its source, the NH+ 4 generated in liver mitochondria is immediately used, together with CO2 (as HCO− 3) produced by mitochondrial respiration, to form carbamoyl phosphate in the matrix (as shown in Fig. 18-10 and explained in detail in Fig. 18-11a). This ATP-dependent reaction is catalyzed by carbamoyl phosphate synthetase I, a regulatory enzyme (see below). The mitochondrial form of the enzyme is distinct from the cytosolic (II) form, which has a separate function in pyrimidine biosynthesis (Chapter 22). MECHANISM FIGURE 18-11 Nitrogen-acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two separate reactions, each requiring ATP. (a) In the first reaction, catalyzed by carbamoyl phosphate synthetase I, the first nitrogen enters from ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activation steps ( and ). (b) In the second reaction, catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. This reaction has two steps. Activation of the ureido oxygen of citrulline in step sets up the addition of aspartate in step . The carbamoyl phosphate, which functions as an activated carbamoyl group donor, now enters the urea cycle. The cycle has only four enzymatic steps. First, carbamoyl phosphate donates its carbamoyl group to ornithine to form citrulline, with the release of Pi (Fig. 18-10, step ). The reaction is catalyzed by ornithine transcarbamoylase. Ornithine is not one of the 20 common amino acids found in proteins, but it is a key intermediate in arginine biosynthesis and nitrogen metabolism in general. It is synthesized from glutamate in a five-step pathway described in Chapter 22. Ornithine plays a role resembling that of oxaloacetate in the citric acid cycle, accepting material at each turn of the urea cycle. The citrulline produced in the first step of the urea cycle passes from the mitochondrion to the cytosol. The next two steps bring in the second amino group, featuring aspartate as the amino group donor. The aspartate is generated in the mitochondria by transamination between glutamate and oxaloacetate, and then transported into the cytosol. A condensation reaction between the amino group of aspartate and the ureido (carbonyl) group of citrulline forms argininosuccinate (step in Fig. 18-10). This cytosolic reaction, catalyzed by argininosuccinate synthetase, requires ATP and proceeds through a citrullyl-AMP intermediate (Fig. 18-11b). The argininosuccinate is then cleaved by argininosuccinase (step in Fig. 18-10) to form free arginine and fumarate, the latter being converted to malate before entering mitochondria to join the pool of citric acid cycle intermediates. This is the only reversible step in the urea cycle. In the last reaction of the urea cycle (step ), the cytosolic enzyme arginase cleaves arginine to yield urea and ornithine. Ornithine is transported into the mitochondrion to initiate another round of the urea cycle. As we noted in Chapter 16, the enzymes of many metabolic pathways are clustered in metabolons (p. 595), with the product of one enzyme reaction being channeled directly to the next enzyme in the pathway. In the urea cycle, the mitochondrial and cytosolic enzymes seem to be clustered in this way. The citrulline transported out of the mitochondrion is not diluted into the general pool of metabolites in the cytosol but is passed directly to the active site of argininosuccinate synthetase. This channeling between enzymes continues for argininosuccinate, arginine, and ornithine. Only urea is released into the general cytosolic pool of metabolites. The Citric Acid and Urea Cycles Can Be Linked The fumarate produced in the argininosuccinase reaction is also an intermediate of the citric acid cycle. Thus, the cycles are, in principle, interconnected — in a process dubbed the “Krebs bicycle” (Fig. 18-12). However, each cycle can operate independently, and communication between them depends on the transport of key intermediates between the mitochondrion and cytosol. Major transporters in the inner mitochondrial membrane include the malate–α -ketoglutarate transporter, the glutamate-aspartate transporter, and the glutamate-OH− transporter. Together, these transporters facilitate the movement of malate and glutamate into the mitochondrial matrix and the movement of aspartate and α -ketoglutarate out to the cytosol. FIGURE 18-12 Links between the urea cycle and citric acid cycle. The interconnected cycles have been called the “Krebs bicycle.” The pathways linking the citric acid and urea cycles are known as the aspartate-argininosuccinate shunt; these effectively link the fates of the amino groups and the carbon skeletons of amino acids. The interconnections are quite elaborate. For example, some citric acid cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol — whether by the urea cycle, purine biosynthesis, or other processes — can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria to enter the citric acid cycle. These processes are further intertwined with the malate-aspartate shuttle, a set of reactions that brings reducing equivalents into the mitochondrion. These different cycles and processes rely on a limited number of transporters in the inner mitochondrial membrane. Several enzymes of the citric acid cycle, including fumarase (fumarate hydratase) and malate dehydrogenase (p. 586), are also present as isozymes in the cytosol. There is no transporter to directly move the fumarate generated in cytosolic arginine synthesis back into the mitochondrial matrix. However, fumarate can be converted to malate in the cytosol. Fumarate and malate can be further metabolized in the cytosol, or malate can be transported into mitochondria for use in the citric acid cycle. Aspartate formed in mitochondria by transamination between oxaloacetate and glutamate can be transported to the cytosol, where it serves as nitrogen donor in the urea cycle reaction catalyzed by argininosuccinate synthetase. These reactions, making up the aspartate-argininosuccinate shunt, provide metabolic links between the separate pathways by which the amino groups and carbon skeletons of amino acids are processed. The use of aspartate as a nitrogen donor in the urea cycle may appear to be a relatively complicated way to introduce the second amino group into urea. However, we shall see in Chapter 22 that this pathway for nitrogen incorporation is one of the two common ways to introduce amino groups into biomolecules. In the urea cycle, additional pathway interconnections can help explain why aspartate is used as a nitrogen donor. The urea and citric acid cycles are closely tied to an additional process that brings NADH, in the form of reducing equivalents, into the mitochondrion. As detailed in the next chapter, the NADH produced by glycolysis, fatty acid oxidation, and other processes cannot be transported across the mitochondrial inner membrane. Reducing equivalents are instead brought into the mitochondrion by converting aspartate to oxaloacetate in the cytosol, reducing the oxaloacetate to malate with NADH, and transporting the malate into the mitochondrial matrix via the malate–α - ketoglutarate transporter. Once inside the mitochondrion, the malate can be reconverted to oxaloacetate while generating NADH. The oxaloacetate is converted to aspartate in the matrix and transported out of the mitochondrion by the aspartate- glutamate transporter. This malate-aspartate shuttle completes yet another cycle that functions to keep the mitochondrion supplied with NADH (Fig. 18-12; see also Fig. 19-31). These processes require that a balance be maintained in the cytosol between the concentrations of glutamate and aspartate. The enzyme that transfers amino groups between these key amino acids is aspartate aminotransferase, AAT (also called glutamate-oxaloacetate transaminase, GOT). This is one of the most active enzymes in hepatocytes and other tissues. When tissue damage occurs, this easily assayed enzyme and others leak into the blood. Thus, measuring blood levels of liver enzymes is important in diagnosing a variety of medical conditions (Box 18- 1). BOX 18-1 MEDICINE Assays for Tissue Damage Analyses of certain enzyme activities in blood serum give valuable diagnostic information for several disease conditions. Blood levels of alanine aminotransferase (ALT; also called glutamate-pyruvate transaminase, GPT) and aspartate aminotransferase (AAT; also called glutamate-oxaloacetate transaminase, GOT) are important in the diagnosis of liver damage, toxicity associated with long-term drug use, or infection. When tissue is damaged, a variety of enzymes, including these aminotransferases, leak from injured cells into the bloodstream. Measurements of the blood serum concentrations of the two aminotransferases by the SGPT and SGOT tests (S for serum) — and of another enzyme, creatine kinase, by the SCK test — can provide information about the severity of the damage. The SGOT and SGPT tests are used in occupational medicine to determine whether people exposed to carbon tetrachloride, chloroform, or other industrial solvents have suffered liver damage. Liver degeneration caused by these solvents is accompanied by leakage of various enzymes from injured hepatocytes into the blood. Aminotransferases are most useful in the monitoring of people exposed to these chemicals, because these enzyme activities are high in liver and thus are likely to be among the proteins leaked from damaged hepatocytes; also, they can be detected in the bloodstream in very small amounts. The Activity of the Urea Cycle Is Regulated at Two Levels The flux of nitrogen through the urea cycle in an individual animal varies with diet. When the dietary intake is primarily protein, the carbon skeletons of amino acids are used for fuel, producing much urea from the excess amino groups. During prolonged starvation, when breakdown of muscle protein begins to supply much of the organism’s metabolic energy, urea production also increases substantially. These changes in demand for urea cycle activity are met over the long term by regulation of the rates of synthesis of the four urea cycle enzymes and carbamoyl phosphate synthetase I in the liver. All five enzymes are synthesized at higher rates in starving animals and in animals on very-high-protein diets than in well- fed animals eating primarily carbohydrates and fats. Animals on protein-free diets produce lower levels of urea cycle enzymes. On a shorter time scale, allosteric regulation of at least one key enzyme adjusts the flux through the urea cycle. The first enzyme in the pathway, carbamoyl phosphate synthetase I, is allosterically activated by N-acetylglutamate, which is synthesized from acetyl-CoA and glutamate by N-acetylglutamate synthase (Fig. 18-13). In plants and microorganisms, this enzyme catalyzes the first step in the de novo synthesis of arginine from glutamate (see Fig. 22-12), but in mammals, N-acetylglutamate synthase activity in the liver has a purely regulatory function (mammals lack the other enzymes needed to convert glutamate to arginine). The steady-state levels of N-acetylglutamate are determined by the concentrations of glutamate and acetyl-CoA (the substrates for N-acetylglutamate synthase) and arginine (an activator of N-acetylglutamate synthase, and thus an activator of the urea cycle). FIGURE 18-13 Synthesis of N-acetylglutamate and its activation of carbamoyl phosphate synthetase I. Pathway Interconnections Reduce the Energetic Cost of Urea Synthesis If we consider the urea cycle in isolation, we see that the synthesis of one molecule of urea requires four high-energy phosphate groups (Fig. 18-10). Two ATP molecules are required to make carbamoyl phosphate, and one ATP to make argininosuccinate — the latter ATP undergoing a pyrophosphate cleavage to AMP and PPi, which is hydrolyzed to two Pi. The overall equation of the urea cycle is However, this apparent cost is compensated for by the pathway interconnections detailed above. The fumarate generated by the urea cycle is converted to malate, and the malate is transported into the mitochondrion (Fig. 18-12). Inside the mitochondrial matrix, NADH is generated in the malate dehydrogenase reaction. Each NADH molecule can generate up to 2.5 ATP during mitochondrial respiration, greatly reducing the overall energetic cost of urea synthesis (mitochondrial respiration is discussed further in Chapter 19). Genetic Defects in the Urea Cycle Can Be Life-Threatening Infants with severe genetic defects in any enzyme involved in urea formation oen appear normal at birth. However, they soon 2NH+ 4 +HCO−3 +3ATP4− +H2O → urea+2ADP3− +4P2− i +AM develop symptoms of hyperammonemia, including cerebral edema, lethargy, and hyperventilation. Without treatment, early death usually results. Symptoms may be less severe in patients retaining partial enzyme activity. These patients cannot tolerate protein-rich diets. Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea and exported into the bloodstream, and, as we have seen, ammonia is highly toxic. Given that most urea cycle steps are irreversible, the absent enzyme activity can oen be identified by determining which cycle intermediate is present in elevated concentration in the blood and/or urine. Although the breakdown of amino acids can have serious health consequences in individuals with urea cycle deficiencies, a protein-free diet is not a treatment option. Humans are incapable of synthesizing half of the 20 common amino acids; they must obtain these essential amino acids in the diet (Table 18-1). TABLE 18-1 Nonessential and Essential Amino Acids for Humans and the Albino Rat Nonessential Conditionally essential Essential Alanine Arginine Histidine Asparagine Cysteine Isoleucine Aspartate Glutamine Leucine Glutamate Glycine Lysine Serine Proline Methionine a Tyrosine Phenylalanine Threonine Tryptophan Valine Required to some degree in young, growing animals and/or sometimes during illness. A variety of treatments are available for individuals with urea cycle defects. Careful administration of the aromatic acids benzoate or phenylbutyrate in the diet can help lower the level of ammonia in the blood. Benzoate is converted to benzoyl-CoA, which combines with glycine to form hippurate (Fig. 18-14, le). The glycine used up in this reaction must be regenerated, and ammonia is thus taken up in the glycine synthase reaction. Phenylbutyrate is converted to phenylacetate by β oxidation. The phenylacetate is then converted to phenylacetyl-CoA, which combines with glutamine to form phenylacetylglutamine (Fig. 18- 14, right). The resulting removal of glutamine triggers its further synthesis by glutamine synthetase (see Eqn 22-1) in a reaction that takes up ammonia. Both hippurate and phenylacetylglutamine are nontoxic compounds that are excreted in the urine. The pathways shown in Figure 18-14 make only minor contributions to normal metabolism, but they become prominent when aromatic acids are ingested. a FIGURE 18-14 Treatment for deficiencies in urea cycle enzymes. The aromatic acids benzoate and phenylbutyrate, administered in the diet, are metabolized and combine with glycine and glutamine, respectively. The products are excreted in the urine. Subsequent synthesis of glycine and glutamine to replenish the pool of these intermediates removes ammonia from the bloodstream. Other therapies are more specific to a particular enzyme deficiency. Deficiency of N-acetylglutamate synthase results in the absence of the normal activator of carbamoyl phosphate synthetase I (Fig. 18-13). This condition can be treated by administering carbamoyl glutamate, an analog of N- acetylglutamate that is effective in activating carbamoyl phosphate synthetase I. Supplementing the diet with arginine is useful in treating deficiencies of ornithine transcarbamoylase, argininosuccinate synthetase, and argininosuccinase. Many of these treatments must be accompanied by strict dietary control and supplements of essential amino acids. In the rare cases of arginase deficiency, arginine, the substrate of the defective enzyme, must be excluded from the diet.

SUMMARY 18.2 Nitrogen Excretion and the Urea Cycle In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine — the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine; ornithine is regenerated in each turn of the cycle. The urea cycle results in a net conversion of oxaloacetate to fumarate, both of which are intermediates in the citric acid cycle. The two cycles are thus interconnected. The activity of the urea cycle is regulated at the level of enzyme synthesis and by allosteric regulation of the enzyme that catalyzes the formation of carbamoyl phosphate. The energetic cost of the urea cycle is reduced by cycle interconnections. Genetic diseases involving urea cycle enzyme deficiencies have serious consequences but sometimes can be managed by dietary intervention. 18.3 Pathways of Amino AcidDegradation Amino acid catabolism normally accounts for only 10% to 15% of the human body’s energy production; these pathways are not nearly as active as glycolysis and fatty acid oxidation. Flux through these catabolic routes also varies greatly, depending on the balance between requirements for biosynthetic processes and the availability of a particular amino acid. The 20 catabolic pathways converge to form only six major products: pyruvate, acetyl-CoA, α -ketoglutarate, succinyl- CoA, fumarate, and oxaloacetate. All of these enter the citric acid cycle (Fig. 18-15). From here the carbon skeletons are diverted to gluconeogenesis or ketogenesis; alternatively, they are completely oxidized as fuel to CO2 and H2O. FIGURE 18-15 Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded by at least two different pathways (see Figs 18-19, 18-27), and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic. We summarize the individual pathways for the 20 amino acids in flow diagrams, each leading to a specific point of entry into the citric acid cycle. In these diagrams, the carbon atoms that enter the citric acid cycle are shown in color. Note that some amino acids appear more than once, reflecting different fates for different parts of their carbon skeletons. Rather than examining every step of every pathway in amino acid catabolism, we single out for special discussion some enzymatic reactions that are particularly noteworthy for their mechanisms or their medical significance. Some Amino Acids Can Contribute to Gluconeogenesis, Others to Ketone Body Formation The seven amino acids that are degraded entirely or in part to acetoacetyl-CoA and/or acetyl-CoA — phenylalanine, tyrosine, isoleucine, leucine, tryptophan, threonine, and lysine — can yield ketone bodies in the liver, where acetoacetyl-CoA is converted to acetoacetate and then to acetone and β -hydroxybutyrate (see Fig. 17- 16). These are the ketogenic amino acids (Fig. 18-15). Their ability to form ketone bodies is particularly evident in uncontrolled diabetes mellitus, in which the liver produces large amounts of ketone bodies from both fatty acids and the ketogenic amino acids. Ketone bodies may also be metabolized in the brain as fuel in place of glucose in cases during starvation. The amino acids that are degraded to pyruvate, α -ketoglutarate, succinyl-CoA, fumarate, and/or oxaloacetate can be converted to glucose and glycogen by pathways described in Chapter 14. They are the glucogenic amino acids. The division between ketogenic and glucogenic amino acids is not sharp; five amino acids — tryptophan, phenylalanine, tyrosine, threonine, and isoleucine — are both ketogenic and glucogenic. All amino acids except for lysine and leucine can make some contribution to gluconeogenesis. Catabolism of amino acids is particularly critical to the survival of animals with high-protein diets or during starvation. Leucine is an exclusively ketogenic amino acid that is very common in proteins. Its degradation makes a substantial contribution to ketosis under starvation conditions. Several Enzyme Cofactors Play Important Roles in Amino Acid Catabolism The structural diversity of amino acids is reflected in the varied reaction types encountered in their breakdown pathways. We begin our study of these pathways by noting important classes of reactions that recur and introducing their enzyme cofactors. We have already considered one important class: transamination reactions requiring pyridoxal phosphate. One-carbon transfers are another common type of reaction in amino acid catabolism. Such transfers usually involve one of three cofactors: biotin, tetrahydrofolate, or S-adenosylmethionine (Fig. 18- 16). These cofactors transfer one-carbon groups in different oxidation states: biotin transfers carbon in its most oxidized state, CO2 (see Fig. 14-17); tetrahydrofolate transfers one-carbon groups in intermediate oxidation states and sometimes as methyl groups; and S- adenosylmethionine transfers methyl groups, the most reduced state of carbon. The latter two cofactors are especially important in amino acid and nucleotide metabolism. FIGURE 18-16 Some enzyme cofactors important in one-carbon transfer reactions. The nitrogen atoms to which one-carbon groups are attached in tetrahydrofolate are shown in blue. Tetrahydrofolate (H 4 folate), synthesized in bacteria, consists of substituted pterin (6-methylpterin), p-aminobenzoate, and glutamate moieties (Fig. 18-16). The oxidized form, folate, is a vitamin for mammals; it is converted in two steps to tetrahydrofolate by the enzyme dihydrofolate reductase. The one-carbon group undergoing transfer, in any of three oxidation states, is bonded to N-5 or N-10 or both. The most reduced form of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group (Fig. 18-17). Most forms of tetrahydrofolate are interconvertible and serve as donors of one-carbon units in a variety of metabolic reactions. The primary source of one-carbon units for tetrahydrofolate is the carbon removed in the conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate. FIGURE 18-17 Conversions of one-carbon units on tetrahydrofolate. The different molecular species are grouped according to oxidation state, with the most reduced at the top and most oxidized at the bottom. All species within a single shaded box are at the same oxidation state. The conversion of N5,N10-methylenetetrahydrofolate to N5-methyltetrahydrofolate is effectively irreversible. The enzymatic transfer of formyl groups, as in purine synthesis (see Fig. 22-35) and in the formation of formylmethionine in bacteria (Chapter 27), generally uses N10- formyltetrahydrofolate rather than N5-formyltetrahydrofolate. The latter species is significantly more stable and therefore a weaker donor of formyl groups. N5-Formyltetrahydrofolate is a minor byproduct of the cyclohydrolase reaction, and can also form spontaneously. Conversion of N5-formyltetrahydrofolate to N5,N10-methenyltetrahydrofolate requires ATP, because of an otherwise unfavorable equilibrium. Note that N5-formiminotetrahydrofolate is derived from histidine in a pathway shown in Figure 18-26. Although tetrahydrofolate can carry a methyl group at N-5, the transfer potential of this methyl group is insufficient for most biosynthetic reactions. S-Adenosylmethionine (adoMet) is the preferred cofactor for biological methyl group transfers. It is synthesized from ATP and methionine by the action of methionine adenosyl transferase (Fig. 18- 18, step ). This reaction is unusual in that the nucleophilic sulfur atom of methionine attacks the 5′ carbon of the ribose moiety of ATP rather than one of the phosphorus atoms. Triphosphate is released and is cleaved to Pi and PPi on the enzyme, and the PPi is cleaved by inorganic pyrophosphatase; thus three bonds, including two bonds of high-energy phosphate groups, are broken in this reaction. The only other known reaction in which triphosphate is displaced from ATP occurs in the synthesis of coenzyme B12 (see Box 17-2, Fig. 3).

FIGURE 18-18 Synthesis of methionine and S-adenosylmethionine in an activated-methyl cycle. The steps are described in the text. In the methionine synthase reaction (step ), the methyl group is transferred to cobalamin to form methylcobalamin, which is the methyl donor in the formation of methionine. S-Adenosylmethionine, which has a positively charged sulfur (and is thus a sulfonium ion), is a powerful methylating agent in several biosynthetic reactions. The methyl group acceptor (step ) is designated R. S-Adenosylmethionine is a potent alkylating agent by virtue of its destabilizing sulfonium ion. The methyl group is subject to attack by nucleophiles and is about 1,000 times more reactive than the methyl group of N5-methyltetrahydrofolate. Transfer of the methyl group from S-adenosylmethionine to an acceptor yields S-adenosylhomocysteine (Fig. 18-18, step ), which is subsequently broken down to homocysteine and adenosine (step ). Methionine is regenerated by transfer of a methyl group to homocysteine in a reaction catalyzed by methionine synthase (step ), and methionine is reconverted to S-adenosylmethionine to complete an activated-methyl cycle. One form of methionine synthase common in bacteria uses N5- methyltetrahydrofolate as a methyl donor. Another form of the enzyme present in some bacteria and mammals uses N5- methyltetrahydrofolate, but the methyl group is first transferred to cobalamin, derived from coenzyme B12, to form methylcobalamin as the methyl donor in methionine formation. This reaction and the rearrangement of L-methylmalonyl-CoA to succinyl-CoA (see Box 17-2, Fig. 1a) are the only known coenzyme B12–dependent reactions in mammals. The vitamins B12 and folate are closely linked in these pathways. The anemia observed in the rare B12 deficiency disease pernicious anemia (Box 17-2) can be traced to the methionine synthase reaction. As noted above, the methyl group of methylcobalamin is derived from N5-methyltetrahydrofolate, and this is the only reaction in mammals that uses N5-methyltetrahydrofolate. The reaction converting the N5,N10-methylene form to the N5-methyl form of tetrahydrofolate is irreversible (Fig. 18-17). Thus, if coenzyme B12 is not available for the synthesis of methylcobalamin, metabolic folates become trapped in the N5-methyl form. The anemia associated with vitamin B12 deficiency is called megaloblastic anemia. It manifests as a decline in the production of mature erythrocytes (red blood cells) and the appearance in the bone marrow of immature precursor cells, or megaloblasts. Erythrocytes are gradually replaced in the blood by smaller numbers of abnormally large erythrocytes called macrocytes. The defect in erythrocyte development is a direct consequence of the depletion of the N5,N10- methylenetetrahydrofolate, which is required for synthesis of the thymidine nucleotides needed for DNA synthesis (see Chapter 22). Folate deficiency, in which all forms of tetrahydrofolate are depleted, also produces anemia, for much the same reasons. The anemia symptoms of B12 deficiency can be alleviated by administering either vitamin B12 or folate. However, it is dangerous to treat pernicious anemia by folate supplementation alone, because the neurological symptoms of B12 deficiency will progress. These symptoms do not arise from the defect in the methionine synthase reaction. Instead, the impaired methylmalonyl-CoA mutase (see Box 17-2 and Fig. 17-12) causes accumulation of unusual, odd-number fatty acids in neuronal membranes. The anemia associated with folate deficiency is thus oen treated by administering both folate and vitamin B12, at least until the metabolic source of the anemia is unambiguously defined. Early diagnosis of B12 deficiency is important because some of its associated neurological conditions may be irreversible. Folate deficiency also reduces the availability of the N5- methyltetrahydrofolate required for methionine synthase function. This leads to a rise in homocysteine levels in blood, a condition linked to heart disease, hypertension, and stroke. High levels of homocysteine may be responsible for 10% of all cases of heart disease. The condition is treated with folate supplements. Tetrahydrobiopterin, another cofactor of amino acid catabolism, is similar to the pterin moiety of tetrahydrofolate, but it is not involved in one-carbon transfers; instead it participates in oxidation reactions. We consider its mode of action when we discuss phenylalanine degradation (see Fig. 18-24). Six Amino Acids Are Degraded to Pyruvate

The carbon skeletons of six amino acids — alanine, tryptophan, cysteine, serine, glycine, and threonine — are converted in whole or in part to pyruvate. The pyruvate can then be converted to acetyl-CoA and eventually oxidized via the citric acid cycle, or to oxaloacetate and shunted into gluconeogenesis (Fig. 18-19). Alanine yields pyruvate directly on transamination with α -ketoglutarate, and the side chain of tryptophan is cleaved to yield alanine and thus pyruvate. Cysteine is converted to pyruvate in two steps; one removes the sulfur atom, the other is a transamination. Serine is converted to pyruvate by serine dehydratase. Both the β -hydroxyl and the α -amino groups of serine are removed in this single pyridoxal phosphate– dependent reaction (Fig. 18-20a). FIGURE 18-19 Catabolic pathways for alanine, tryptophan, cysteine, serine, glycine, and threonine. Carbon atoms here and in subsequent figures are color-coded as necessary to trace their fates. The fate of the indole group of tryptophan is shown in Figure 18-21. Details of most of the reactions involving serine and glycine are shown in Figure 18-20. Several pathways for cysteine degradation lead to pyruvate. The pathway for threonine degradation shown here accounts for only about a third of threonine catabolism (for the alternative pathway, see Fig. 18- 27). MECHANISM FIGURE 18-20 Interplay of the pyridoxal phosphate and tetrahydrofolate cofactors in serine and glycine metabolism. The first step in each of these reactions (not shown) involves the formation of a covalent imine linkage between enzyme-bound PLP and the substrate amino acid — serine in (a), glycine in (b) and (c). (a) A PLP-catalyzed elimination of water in the serine dehydratase reaction (step ) begins the pathway to pyruvate. (b) In the serine hydroxymethyltransferase reaction, a PLP-stabilized carbanion (product of step ) is a key intermediate in the reversible transfer of the methylene group (as—CH2—OH) from N5,N10- methylenetetrahydrofolate to form serine. (c) The glycine cleavage enzyme is a multienzyme complex, with components P, H, T, and L. The overall reaction, which is reversible, converts glycine to CO2 and NH+4, with the second glycine carbon taken up by tetrahydrofolate to form N5,N10-methylenetetrahydrofolate. Pyridoxal phosphate activates the α carbon of amino acids at critical stages in all these reactions, and tetrahydrofolate carries a one-carbon unit in two of them (see Figs 18-6, 18-17). Glycine is degraded via three pathways, only one of which leads to pyruvate. Glycine is converted to serine by enzymatic addition of a hydroxymethyl group (Fig. 18-19, 18-20b). This reaction, catalyzed by serine hydroxymethyltransferase, requires the coenzymes tetrahydrofolate and pyridoxal phosphate. The serine is converted to pyruvate as described above. In the second pathway, which predominates in animals, glycine undergoes oxidative cleavage to CO2, NH+4, and a methylene group (—CH2—) (Figs. 18-19, 18-20c). This readily reversible reaction, catalyzed by glycine cleavage enzyme (also called glycine synthase), also requires tetrahydrofolate, which accepts the methylene group. In this oxidative cleavage pathway, the two carbon atoms of glycine do not enter the citric acid cycle. One carbon is lost as CO2 and the other becomes the methylene group of N5,N10- methylenetetrahydrofolate (Fig. 18-17), a one-carbon group donor in certain biosynthetic pathways. This second pathway for glycine degradation seems to be critical in mammals. Humans with serious defects in glycine cleavage enzyme activity suffer from a condition known as nonketotic hyperglycinemia. The condition is characterized by elevated serum levels of glycine, leading to severe intellectual disability and death in very early childhood. At high levels, glycine is an inhibitory neurotransmitter, which may explain the neurological effects of the disease. Perhaps more important, high levels of glycine increase the levels of 2-amino-3- ketobutyrate, an unstable intermediate in the degradation of threonine in mitochondria (Fig. 18-19). 2-Amino-3-ketobutyrate decarboxylates spontaneously to form the toxic metabolite aminoacetone, which is readily metabolized to the highly reactive methylglyoxal, a molecule that modifies both protein and DNA. Methylglyoxal is also a byproduct of glycolysis and is implicated in the progression of type 2 diabetes (Box 7-2). Many genetic defects of amino acid metabolism have been identified in humans (Table 18-2). We shall encounter several more in this chapter. TABLE 18-2 Some Human Genetic Disorders Affecting Amino Acid Catabolism Medical condition Approximate incidence (per 100,000 births) Defective process Defective enzyme Symptoms and effects Albinism <3 Melanin synthesis from tyrosine Tyrosine 3- monooxygenase (tyrosinase) Lack of pigmentation; white hair, pink skin Alkaptonuria <0.4 Tyrosine degradation Homogentisate 1,2-dioxygenase Dark pigment in urine; late- developing arthritis Argininemia <0.5 Urea synthesis Arginase Intellectual disability Argininosuccinic acidemia <1.5 Urea synthesis Argininosuccinase Vomiting; convulsions Carbamoyl phosphate synthetase I deficiency <0.5 Urea synthesis Carbamoyl phosphate synthetase I Lethargy; convulsions; early death Homocystinuria <0.5 Methionine degradation Cystathionine β - synthase Faulty bone development; intellectual disability Maple syrup urine disease (branched-chain ketoaciduria) <0.4 Isoleucine, leucine, and valine degradation Branched-chain α -keto acid dehydrogenase complex Vomiting; convulsions; intellectual disability; early death Methylmalonic acidemia <0.5 Conversion of propionyl-CoA to succinyl- CoA Methylmalonyl- CoA mutase Vomiting; convulsions; intellectual disability; early death Phenylketonuria <8 Conversion of phenylalanine to tyrosine Phenylalanine hydroxylase Neonatal vomiting; intellectual disability In the third and final pathway of glycine degradation, the achiral glycine molecule is a substrate for the enzyme D-amino acid oxidase. The glycine is converted to glyoxylate, an alternative substrate for lactate dehydrogenase (p. 526). Glyoxylate is oxidized in an NAD+- dependent reaction to oxalate: The function of D-amino acid oxidase, present at high levels in the kidney, is thought to be the detoxification of ingested D-amino acids derived from bacterial cell walls and from grilled foodstuffs (high heat causes some spontaneous racemization of the L-amino acids in proteins). Oxalate, whether obtained in foods or produced enzymatically in the kidneys, has medical significance. Crystals of calcium oxalate account for up to 75% of all kidney stones. There are two significant pathways for threonine degradation. One pathway leads to pyruvate via glycine (Fig. 18-19). The conversion to glycine occurs in two steps, with threonine first converted to 2-amino-3- ketobutyrate by the action of threonine dehydrogenase. This pathway is important in a few classes of rapidly dividing human cells, such as embryonic stem cells. The glycine generated by this pathway is broken down primarily by the glycine cleavage enzyme (Fig. 18-19). The N5,N10 -methylenetetrahydrofolate thus generated (Fig. 18-20c) is needed for the synthesis, via pathways described in Chapter 22, of nucleotides used in DNA replication. However, in most human tissues, the degradation of threonine via glycine is a relatively minor pathway, accounting for 10% to 30% of threonine catabolism. It is more important in some other mammals. The major pathway in most human tissues leads to succinyl- CoA, as described later in this chapter. Seven Amino Acids Are Degraded to Acetyl-CoA

Portions of the carbon skeletons of seven amino acids — tryptophan, lysine, phenylalanine, tyrosine, leucine, isoleucine, and threonine — yield acetyl-CoA and/or acetoacetyl-CoA, the latter being converted to acetyl-CoA (Fig. 18-21). Some of the final steps in the degradative pathways for leucine, lysine, and tryptophan resemble steps in the oxidation of fatty acids (see Fig. 17-9). Threonine (not shown in Fig. 18-21) yields some acetyl-CoA via the minor pathway illustrated in Figure 18-19. FIGURE 18-21 Catabolic pathways for tryptophan, lysine, phenylalanine, tyrosine, leucine, and isoleucine. These amino acids donate some of their carbons (red) to acetoacetate and acetyl- CoA. In the pathway for leucine catabolism, the carbon in acetoacetate that is donated by CO2 is surrounded by a box to distinguish it from the three carbons that come from leucine itself. Tryptophan, phenylalanine, tyrosine, and isoleucine also contribute carbons (blue) to pyruvate or citric acid cycle intermediates. The phenylalanine pathway is described in more detail in Figure 18-23. The fate of nitrogen atoms is not traced in this scheme; in most cases they are transferred to α -ketoglutarate to form glutamate. The degradative pathways of two of these seven amino acids deserve special mention. Tryptophan breakdown is the most complex of all the pathways of amino acid catabolism in animal tissues; portions of tryptophan (four of its carbons) yield acetyl-CoA via acetoacetyl-CoA. Some of the intermediates in tryptophan catabolism are precursors for the synthesis of other biomolecules (Fig. 18-22), including nicotinate, a precursor of NAD and NADP in animals; serotonin, a neurotransmitter in vertebrates; and indoleacetate, a growth factor in plants. Some of these biosynthetic pathways are described in more detail in Chapter 22 (see Figs. 22-30, 22-31). FIGURE 18-22 Tryptophan as precursor. The aromatic rings of tryptophan give rise to nicotinate (niacin), indoleacetate, and serotonin. Colored atoms trace the source of the ring atoms in nicotinate. The breakdown of phenylalanine is noteworthy because genetic defects in the enzymes of this pathway lead to several inheritable human diseases (Fig. 18-23), as discussed below. Phenylalanine and its oxidation product tyrosine (both with nine carbons) are degraded into two fragments, both of which can enter the citric acid cycle: four of the nine carbon atoms yield free acetoacetate, which is converted to acetoacetyl-CoA and thus acetyl-CoA, and a second four-carbon fragment is recovered as fumarate. Eight of the nine carbons of these two amino acids thus enter the citric acid cycle; the remaining carbon is lost as CO2. Phenylalanine, aer its hydroxylation to tyrosine, is also the precursor of dopamine, a neurotransmitter, and of norepinephrine and epinephrine, hormones secreted by the adrenal medulla (see Fig. 22- 31). Melanin, the black pigment of skin and hair, is also derived from tyrosine. FIGURE 18-23 Catabolic pathways for phenylalanine and tyrosine. In humans these amino acids are normally converted to acetoacetyl-CoA and fumarate. Genetic defects in many of these enzymes cause inheritable human diseases (shaded yellow). Phenylalanine Catabolism Is Genetically Defective in Some People Given that many amino acids are either neurotransmitters or precursors or antagonists of neurotransmitters, it is not surprising that genetic defects of amino acid metabolism can cause defective neural development and intellectual deficits. In most such diseases, specific intermediates accumulate. For example, a genetic defect in phenylalanine hydroxylase, the first enzyme in the catabolic pathway for phenylalanine (Fig. 18-23), is responsible for the disease phenylketonuria (PKU), the most common cause of elevated levels of phenylalanine in the blood (hyperphenylalaninemia). Phenylalanine hydroxylase (also called phenylalanine-4- monooxygenase) is one of a general class of enzymes called mixed- function oxygenases (see Box 21-1), all of which catalyze simultaneous hydroxylation of a substrate by an oxygen atom of O2 and reduction of the other oxygen atom to H2O. Phenylalanine hydroxylase requires the cofactor tetrahydrobiopterin, which carries electrons from NADPH to O2 and becomes oxidized to dihydrobiopterin in the process (Fig. 18-24). It is subsequently reduced by the enzyme dihydrobiopterin reductase in a reaction that requires NADPH. FIGURE 18-24 Role of tetrahydrobiopterin in the phenylalanine hydroxylase reaction. The H atom shaded pink is transferred directly from C-4 to C-3 in the reaction. This feature, discovered at the National Institutes of Health, is called the NIH shi . In individuals with PKU, a secondary, normally little-used pathway of phenylalanine metabolism comes into play. In this pathway phenylalanine undergoes transamination with pyruvate to yield phenylpyruvate (Fig. 18-25). Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are excreted in the urine — hence the name “phenylketonuria.” Much of the phenylpyruvate, rather than being excreted as such, is either decarboxylated to phenylacetate or reduced to phenyllactate. Phenylacetate imparts a characteristic honeylike odor to the urine, which nurses have traditionally used to detect PKU in infants. The accumulation of phenylalanine or its metabolites in early life impairs normal development of the brain, causing severe intellectual deficits. This may be caused by excess phenylalanine competing with other amino acids for transport across the blood-brain barrier, resulting in a deficit of required metabolites. FIGURE 18-25 Alternative pathways for catabolism of phenylalanine in phenylketonuria. In PKU, phenylpyruvate accumulates in the tissues, blood, and urine. The urine may also contain phenylacetate and phenyllactate. Phenylketonuria was among the first inheritable metabolic defects discovered in humans. When this condition is recognized early in infancy, intellectual disability can be prevented by rigid dietary control. The diet must supply only enough phenylalanine and tyrosine to meet the needs for protein synthesis. Consumption of protein-rich foods must be curtailed. Natural proteins, such as casein of milk, must first be hydrolyzed and much of the phenylalanine removed to provide an appropriate diet, at least through childhood. Because the artificial sweetener aspartame is a dipeptide of aspartate and the methyl ester of phenylalanine (see Fig. 1-23a), foods sweetened with aspartame bear warnings addressed to individuals on phenylalanine-controlled diets. The dietary restrictions are difficult to follow perfectly for a lifetime, and thus oen do not completely eliminate neurological symptoms. An enzyme substitution treatment has been developed in which the enzyme phenylalanine ammonia lyase is modified with polyethylene glycol (PEGylated) and injected subcutaneously to degrade phenylalanine in proteins ingested as part of a somewhat less restricted diet. Derived from plants, bacteria, and many yeast and fungi, phenylalanine ammonia lyase normally contributes to the biosynthesis of polyphenol compounds such as flavonoids. It degrades phenylalanine to the harmless metabolite trans-cinnamic acid and ammonia; the small amounts of ammonia generated are not toxic. The treatment was approved for patients in 2018. The long-term effects of the treatment continue to be studied. Phenylketonuria can also be caused by a defect in the enzyme that catalyzes the regeneration of tetrahydrobiopterin (Fig. 18-24). The treatment in this case is more complex than restricting the intake of phenylalanine and tyrosine. Tetrahydrobiopterin is also required for the formation of L-3,4-dihydroxyphenylalanine (L-dopa) and 5- hydroxytryptophan — precursors of the neurotransmitters norepinephrine and serotonin, respectively. In phenylketonuria of this type, these precursors must be supplied in the diet, along with tetrahydrobiopterin. Screening newborns for genetic diseases can be highly cost-effective, especially in the case of PKU. The tests (no longer relying on urine odor) are relatively inexpensive, and the detection and early treatment of PKU in infants (eight to ten cases per 100,000 newborns) saves millions of dollars in later health care costs each year. More importantly, the emotional trauma avoided by early detection with these simple tests is inestimable. Another inheritable disease of phenylalanine catabolism is alkaptonuria, in which the defective enzyme is homogentisate dioxygenase (Fig. 18-23). Less serious than PKU, this condition produces few ill effects, although large amounts of homogentisate are excreted and its oxidation turns the urine black. Individuals with alkaptonuria are also prone to develop a form of arthritis. Alkaptonuria is of considerable historical interest. Archibald Garrod discovered in the early 1900s that this condition is inherited, and he traced the cause to the absence of a single enzyme. Garrod was the first to make a connection between an inheritable trait and an enzyme — a great advance on the path that ultimately led to our current understanding of genes and the information pathways described in Part III. Five Amino Acids Are Converted to α - Ketoglutarate The carbon skeletons of five amino acids (proline, glutamate, glutamine, arginine, and histidine) enter the citric acid cycle as α - ketoglutarate (Fig. 18-26). Proline, glutamate, and glutamine have five- carbon skeletons. The cyclic structure of proline is opened by oxidation of the carbon most distant from the carboxyl group to create a Schiff base, then hydrolysis of the Schiff base to form a linear semialdehyde, glutamate γ -semialdehyde. This intermediate is further oxidized at the same carbon to produce glutamate. The action of glutaminase, or any of several enzyme reactions in which glutamine donates its amide nitrogen to an acceptor, converts glutamine to glutamate. Transamination or deamination of glutamate produces α - ketoglutarate. FIGURE 18-26 Catabolic pathways for arginine, histidine, glutamate, glutamine, and proline. These amino acids are converted to α -ketoglutarate. The numbered steps in the histidine pathway are catalyzed by histidine ammonia lyase, urocanate hydratase, imidazolonepropionase, and glutamate formimino transferase. Arginine and histidine contain five adjacent carbons and a sixth carbon attached through a nitrogen atom. The catabolic conversion of these amino acids to glutamate is therefore slightly more complex than the path from proline or glutamine (Fig. 18-26). Arginine is converted to the five-carbon skeleton of ornithine in the urea cycle (Fig. 18-10), and the ornithine is transaminated to glutamate γ -semialdehyde. Conversion of histidine to the five-carbon glutamate occurs in a multistep pathway; the extra carbon is removed in a step that uses tetrahydrofolate as cofactor. Four Amino Acids Are Converted to Succinyl-CoA The carbon skeletons of methionine, isoleucine, threonine, and valine are degraded by pathways that yield succinyl-CoA (Fig. 18-27), an intermediate of the citric acid cycle. Methionine donates its methyl group to one of several possible acceptors through S-adenosyl methionine, and three of its four remaining carbon atoms are converted to the propionate of propionyl-CoA, a precursor of succinyl- CoA. Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting α -keto acid. The remaining five- carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA. Valine undergoes transamination and decarboxylation, then a series of oxidation reactions that convert the remaining four carbons to propionyl-CoA. Some parts of the valine and isoleucine degradative pathways closely parallel steps in fatty acid degradation (see Fig. 17-9). In human tissues, threonine is also converted in two steps to propionyl- CoA. This is the primary pathway for threonine degradation in humans (see Fig. 18-19 for the alternative pathway). The mechanism of the first step is analogous to that catalyzed by serine dehydratase, and the serine and threonine dehydratases may actually be the same enzyme. FIGURE 18-27 Catabolic pathways for methionine, isoleucine, threonine, and valine. These amino acids are converted to succinyl-CoA; isoleucine also contributes two of its carbon atoms to acetyl-CoA (see Fig. 18-21). The pathway of threonine degradation shown here occurs in humans; a pathway found in other organisms is shown in Figure 18-19. The route from methionine to homocysteine is described in more detail in Figure 18-18; the conversion of homocysteine to α - ketobutyrate, in Figure 22-16; and the conversion of propionyl-CoA to succinyl-CoA, in Figure 17- 12. The propionyl-CoA derived from these three amino acids is converted to succinyl-CoA by a pathway described in Chapter 17: carboxylation to methylmalonyl-CoA, epimerization of the methylmalonyl-CoA, and conversion to succinyl-CoA by the coenzyme B12–dependent methylmalonyl-CoA mutase (see Fig. 17-12). In the rare genetic disease known as methylmalonic acidemia, methylmalonyl-CoA mutase is lacking — with serious metabolic consequences (Table 18-2; Box 18-2). BOX 18-2 MEDICINE MMA: Sometimes More than a Genetic Disease Without treatment, an infant born with a genetic deficiency involving the enzyme methylmalonyl-CoA mutase is subject to severe symptoms ranging from seizures and vomiting to lethargy, developmental disease, progressive encephalopathy (brain disease), and early death. A rare and recessive genetic disorder, the condition, methylmalonic acidemia or MMA, affects about 1 child in 48,000 (Fig. 1). For most parents of a child with MMA, the condition brings trials and some heartbreak. For Patricia Stallings, the condition of her undiagnosed child led to much worse. Biochemistry eventually came to the rescue. FIGURE 1 Children with a mutation (red X) that inactivates the enzyme methylmalonyl-CoA mutase cannot degrade isoleucine, methionine, threonine, and valine normally. Instead, a potentially fatal accumulation of methylmalonic acid occurs, with symptoms similar to those of ethylene glycol poisoning. In the summer of 1989, Stallings took her baby son Ryan to the emergency room at the Cardinal Glennon Children’s Hospital in St. Louis. To the attending physician, a toxicologist, Ryan’s fit of vomiting and labored breathing suggested ingestion of the antifreeze ingredient ethylene glycol. The toxicologist suspected foul play and alerted authorities. Analysis of Ryan’s milk bottles by two laboratories seemed to confirm the physician’s fears. Ryan was placed in foster care as soon as he recovered. Unfortunately for both Ryan and his mother, Ryan subsequently died immediately a er a visit from Patricia. Patricia was arrested and charged with murder. While awaiting trial, Patricia learned that she was again pregnant. She gave birth to a second son, David, who was placed in foster care. David developed symptoms similar to Ryan’s and was diagnosed with MMA. Although the symptoms of MMA can mimic those observed in cases of ethylene glycol poisoning, the revelation did not help Patricia. The information about David’s diagnosis was excluded at her trial for the murder of Ryan. In 1991, Patricia Stallings was sentenced to prison for life. The situation was ultimately resolved when William Sly, chair of the Department of Biochemistry and Molecular Biology at St. Louis University, became interested in the case. Working with metabolic disease experts James Shoemaker and Piero Rinaldo, he had new analyses performed on Ryan’s blood and the original milk bottles. The blood showed high levels of methylmalonic acid and ketones, diagnostic of MMA. Surprisingly, unlike the earlier lab reports, they found no ethylene glycol in any of the samples. Could both a hospital lab and a commercial lab have been mistaken? What the biochemists saw provided the final breakthrough. The biochemists presented all of their materials to the Missouri state prosecutor who had handled the case, George McElroy. Both of the original lab analyses had been so unusually shoddy that they were quickly dismissed. Patricia Stallings was cleared of charges and released on September 20, 1991. Branched-Chain Amino Acids Are Not Degraded in the Liver Although much of the catabolism of amino acids takes place in the liver, the three amino acids with branched side chains (leucine, isoleucine, and valine) are oxidized as fuels primarily in muscle, adipose, kidney, and brain tissue. These extrahepatic tissues contain an aminotransferase, absent in liver, that acts on all three branched-chain amino acids to produce the corresponding α -keto acids (Fig. 18-28). The branched-chain α -keto acid dehydrogenase complex then catalyzes oxidative decarboxylation of all three α -keto acids, in each case releasing the carboxyl group as CO2 and producing the acyl-CoA derivative. This reaction is formally analogous to two other oxidative decarboxylations encountered in Chapter 16: oxidation of pyruvate to acetyl-CoA by the pyruvate dehydrogenase complex and oxidation of α - ketoglutarate to succinyl-CoA by the α -ketoglutarate dehydrogenase complex (see Fig. 16-12). In fact, all three enzyme complexes are similar in structure and share essentially the same reaction mechanism. Five cofactors (thiamine pyrophosphate, FAD, NAD, lipoate, and coenzyme A) participate, and the three proteins in each complex catalyze homologous reactions. This is clearly a case in which enzymatic machinery that evolved to catalyze one reaction was “borrowed” by gene duplication and further evolved to catalyze similar reactions in other pathways. FIGURE 18-28 Catabolic pathways for the three branched-chain amino acids: valine, isoleucine, and leucine. All three pathways occur in extrahepatic tissues and share the first two enzymes, as shown here. The branched-chain α -keto acid dehydrogenase complex is analogous to the pyruvate and α -ketoglutarate dehydrogenase complexes and requires the same five cofactors (some not shown here). This enzyme is defective in people with maple syrup urine disease. Experiments with rats have shown that the branched-chain α -keto acid dehydrogenase complex is regulated by covalent modification in response to the content of branched-chain amino acids in the diet. With little or no excess dietary intake of branched-chain amino acids, the enzyme complex is phosphorylated by a protein kinase and thereby inactivated. Addition of excess branched-chain amino acids to the diet results in dephosphorylation and consequent activation of the enzyme. Recall that the pyruvate dehydrogenase complex is subject to similar regulation by phosphorylation and dephosphorylation (see Fig. 16-19). There is a relatively rare genetic disease in which the three branched-chain α -keto acids (as well as their precursor amino acids, especially leucine) accumulate in the blood and “spill over” into the urine. This condition, called maple syrup urine disease because of the characteristic odor imparted to the urine by the α -keto acids, results from a defective branched-chain α -keto acid dehydrogenase complex. Untreated, the disease results in abnormal development of the brain and death in early infancy. Treatment entails rigid control of the diet, limiting the intake of valine, isoleucine, and leucine to the minimum required to permit normal growth. Asparagine and Aspartate Are Degraded to Oxaloacetate The carbon skeletons of asparagine and aspartate ultimately enter the citric acid cycle as malate in mammals or oxaloacetate in bacteria. The enzyme asparaginase catalyzes the hydrolysis of asparagine to aspartate, which undergoes transamination with α -ketoglutarate to yield glutamate and oxaloacetate (Fig. 18-29). The oxaloacetate is converted to malate in the cytosol and then transported into the mitochondrial matrix through the malate–α -ketoglutarate transporter. In bacteria, the oxaloacetate produced in the transamination reaction can be used directly in the citric acid cycle. FIGURE 18-29 Catabolic pathway for asparagine and aspartate. Both amino acids are converted to oxaloacetate. We have now seen how the 20 common amino acids, aer losing their nitrogen atoms, are degraded by dehydrogenation, decarboxylation, and other reactions to yield portions of their carbon backbones in the form of six central metabolites that can enter the citric acid cycle. Those portions degraded to acetyl-CoA are completely oxidized to carbon dioxide and water, with generation of ATP by oxidative phosphorylation. Those portions degraded to other citric acid cycle intermediates can either be oxidized or contribute to gluconeogenesis, depending on the metabolic state. As was the case for carbohydrates and lipids, the degradation of amino acids results ultimately in the generation of reducing equivalents (NADH and FADH2) through the action of the citric acid cycle. Our survey of catabolic processes concludes in the next chapter with a discussion of respiration, in which these reducing equivalents fuel the ultimate oxidative and energy-generating process in aerobic organisms. SUMMARY 18.3 Pathways of Amino Acid Degradation Aer the removal of amino groups, the carbon skeletons of amino acids undergo oxidation to compounds that can enter the citric acid cycle. The citric acid cycle products can be oxidized as fuel. Alternatively, depending on their degradative end product, some amino acids can be converted to ketone bodies, some to glucose, and some to both. Thus, amino acid degradation is integrated into intermediary metabolism and can be critical to survival under conditions in which amino acids are a significant source of metabolic energy. The reactions of these pathways require several cofactors, including tetrahydrofolate and S-adenosylmethionine in one-carbon transfer reactions and tetrahydrobiopterin in the oxidation of phenylalanine by phenylalanine hydroxylase. Alanine, cysteine, glycine, serine, threonine, and tryptophan are converted in whole or in part to pyruvate. Leucine, lysine, phenylalanine, tyrosine, and tryptophan yield acetyl- CoA via acetoacetyl-CoA. Isoleucine, leucine, threonine, and tryptophan also form acetyl-CoA directly. Leucine and lysine are the only amino acids that cannot contribute to gluconeogenesis. Four carbon atoms of phenylalanine and tyrosine give rise to fumarate. Many human genetic diseases are traced to deficiencies in enzymes catalyzing steps in amino acid degradation. Two well-studied examples, phenylketonuria and alkaptonuria, feature defects in phenylalanine degradation. Most amino acid degradation deficiencies are treated with dietary intervention. Arginine, glutamate, glutamine, histidine, and proline are degraded to α -ketoglutarate. Isoleucine, methionine, threonine, and valine produce succinyl-CoA. The branched-chain amino acids (isoleucine, leucine, and valine), unlike the other amino acids, are degraded only in extrahepatic tissues. Asparagine and aspartate produce oxaloacetate. Chapter Review KEY TERMS Terms in bold are defined in the glossary. ammonotelic ureotelic uricotelic aminotransferases transaminases transamination pyridoxal phosphate (PLP) oxidative deamination L-glutamate dehydrogenase glutamine synthetase glutaminase glucose-alanine cycle urea cycle urea creatine kinase essential amino acids ketogenic glucogenic tetrahydrofolate S-adenosylmethionine (adoMet) tetrahydrobiopterin phenylketonuria (PKU) mixed-function oxygenases alkaptonuria maple syrup urine disease PROBLEMS 1. Products of Amino Acid Transamination Name and draw the structure of the α -keto acid resulting when each of the four amino acids listed undergoes transamination with α - ketoglutarate: (a) aspartate, (b) glutamate, (c) alanine, (d) phenylalanine. 2. Measurement of Alanine Aminotransferase Activity The measurement of alanine aminotransferase activity (reaction rate) usually includes an excess of pure lactate dehydrogenase and NADH in the reaction system. The rate of alanine disappearance is equal to the rate of NADH disappearance measured spectrophotometrically. Explain how this assay works. 3. Alanine and Glutamine in the Blood Normal human blood plasma contains all the amino acids required for the synthesis of body proteins, but not in equal concentrations. Alanine and glutamine are present in much higher concentrations than any other amino acids. Suggest why. 4. Glutamate Dehydrogenase Function Increases in ATP levels in blood trigger insulin release by the pancreas, which in turn stimulates the uptake of blood glucose (see Chapter 23, p. 860). Knowing this, suggest why a mutation that prevents inhibition of glutamate dehydrogenase by GTP results in insulin release and hypoglycemia. 5. Distribution of Amino Nitrogen If your diet is rich in alanine but deficient in aspartate, will you show signs of aspartate deficiency? Explain. 6. Lactate versus Alanine as Metabolic Fuel: The Cost of Nitrogen Removal The three carbons in lactate and alanine have identical oxidation states, and animals can use either carbon source as a metabolic fuel. Compare the net ATP yield (moles of ATP per mole of substrate) for the complete oxidation (to CO2 and H2O) of lactate versus alanine when the cost of nitrogen excretion as urea is included.

7. Ammonia Toxicity Resulting from an Arginine-Deficient Diet In a study, cats were fasted overnight then given a single meal complete in all amino acids except arginine. Within 2 hours, blood ammonia levels increased from a normal level of 18μg/L to 140μg/L, and the cats showed the clinical symptoms of ammonia toxicity. A control group fed a complete amino acid diet or an amino acid diet in which arginine was replaced by ornithine showed no unusual clinical symptoms. a. What was the role of fasting in the experiment? b. What caused the ammonia levels to rise in the experimental group? Why did the absence of arginine lead to ammonia toxicity? Is arginine an essential amino acid in cats? Why or why not? c. Why can ornithine be substituted for arginine? 8. Oxidation of Glutamate Write a series of balanced equations and an overall equation for the net reaction describing the oxidation of 2 mol of glutamate to 2 mol of α - ketoglutarate and 1 mol of urea. 9. Transamination and the Urea Cycle Aspartate aminotransferase has the highest activity of all the mammalian liver aminotransferases. Why? 10. The Case against the Liquid Protein Diet A weight- reducing diet heavily promoted some years ago required the daily intake of a “liquid protein” soup made of hydrolyzed gelatin (derived from collagen), water, and an assortment of vitamins. All other food and drink were to be avoided. People on this diet typically lost 10 to 14 lb in the first week. a. Opponents argued that the weight loss was almost entirely due to water loss and would be regained very soon aer a normal diet was resumed. What is the biochemical basis for this argument? b. A few people on this diet died. What are some of the dangers inherent in the diet, and how can they lead to death? 11. Ketogenic Amino Acids Which amino acids are exclusively ketogenic? 12. A Genetic Defect in Amino Acid Metabolism: A Case History A two-year-old child was taken to the hospital. His mother said that he vomited frequently, especially aer feedings. The child’s weight and physical development were below normal. His hair, although dark, contained patches of white. A urine sample treated with ferric chloride (FeCl3) gave a green color characteristic of the presence of phenylpyruvate. Quantitative analysis of urine samples gave the results shown in the table. Concentration (mM) Substance Patient’s urine Normal urine Phenylalanine 7.0 0.01 Phenylpyruvate 4.8 0 Phenyllactate 10.3 0 a. Suggest which enzyme might be deficient in this child. Propose a treatment. b. Why does phenylalanine appear in the urine in large amounts? c. What is the source of phenylpyruvate and phenyllactate? Why does this pathway (normally not functional) come into play when the concentration of phenylalanine rises? d. Why does the boy’s hair contain patches of white? 13. Role of Cobalamin in Amino Acid Catabolism Pernicious anemia is caused by impaired absorption of vitamin B12. What is the effect of this impairment on the catabolism of amino acids? Are all amino acids equally affected? (Hint: See Box 17-2.) 14. Vegetarian Diets Vegetarian diets can provide high levels of antioxidants and a lipid profile that can help prevent coronary disease. However, there can be some associated problems. Blood samples were taken from a large group of volunteer subjects who were vegans (strict vegetarians: no animal products), lactovegetarians (vegetarians who eat dairy products), or omnivores (individuals with a varied diet, including meat). In each case, the volunteers had followed the diet for several years. The blood levels of both homocysteine and methylmalonate were elevated in the vegan group, somewhat lower in the lactovegetarian group, and much lower in the omnivore group. Explain. 15. Pernicious Anemia Vitamin B12 deficiency can arise from a few rare genetic diseases that lead to low B12 levels despite a normal diet that includes B12-rich meat and dairy sources. These conditions cannot be treated with dietary B12 supplements. Explain. 16. Pyridoxal Phosphate Reaction Mechanisms Threonine can be broken down by the enzyme threonine dehydratase, which catalyzes the conversion of threonine to α - ketobutyrate and ammonia. The enzyme uses PLP as a cofactor. Suggest a mechanism for this reaction, based on the mechanisms in Figure 18-6. Note that this reaction includes an elimination at the β carbon of threonine. 17. Pathway of Carbon and Nitrogen in Glutamate Metabolism When [2−14C,15N]glutamate undergoes oxidative degradation in the liver of a rat, in which atoms of the following metabolites will each isotope be found? a. urea b. succinate c. arginine d. citrulline e. ornithine f. aspartate 18. Chemical Strategy of Isoleucine Catabolism Isoleucine is degraded in six steps to propionyl-CoA and acetyl-CoA. a. The chemical process of isoleucine degradation includes strategies analogous to those used in the citric acid cycle and the β oxidation of fatty acids. The intermediates of isoleucine degradation (I to V) shown below are not in the proper order. Use your knowledge and understanding of the citric acid cycle and β -oxidation pathway to arrange the intermediates in the proper metabolic sequence for isoleucine degradation. b. For each step you propose, describe the chemical process, provide an analogous example from the citric acid cycle or β -oxidation pathway (where possible), and indicate any necessary cofactors. 19. Role of Pyridoxal Phosphate in Glycine Metabolism The enzyme serine hydroxymethyltransferase requires pyridoxal phosphate as a cofactor. Propose a mechanism for the reaction catalyzed by this enzyme, in the direction of serine degradation (glycine production). (Hint: See Figs 18-19 and 18-20b.) 20. Parallel Pathways for Amino Acid and Fatty Acid Degradation The carbon skeleton of leucine is degraded by a series of reactions closely analogous to those of the citric acid cycle and β oxidation. For each reaction, (a) through (f), shown below, indicate its type, provide an analogous example from the citric acid cycle or β -oxidation pathway (where possible), and note any necessary cofactors.

21. Treatments for a Genetic Disease The strict dietary controls required to stem the progress of maple syrup urine disease are difficult to follow for a lifetime, and patients may experience poor metabolic control that leads to neurological symptoms. In these cases, treatment can involve an organ transplant from a suitable donor. Organ transplantation involves considerable risk, but success can greatly alleviate this metabolic disorder and reduce the need for dietary restrictions. Which organ could be transplanted to gain this effect, and why? DATA ANALYSIS PROBLEM 22. Maple Syrup Urine Disease Figure 18-28 shows the pathway for the degradation of branched-chain amino acids and the site of the biochemical defect that causes maple syrup urine disease. The initial findings that eventually led to the discovery of the defect in this disease were presented in three papers published in the late 1950s and early 1960s. This problem traces the history of the findings from initial clinical observations to proposal of a biochemical mechanism. Menkes, Hurst, and Craig (1954) presented the cases of four siblings, all of whom died following a similar course of symptoms. In all four cases, the mother’s pregnancy and the birth had been normal. The first 3 to 5 days of each child’s life were also normal. But soon thereaer each child began having convulsions, and the children died between the ages of 11 days and 3 months. Autopsy showed considerable swelling of the brain in all cases. The children’s urine had a strong, unusual “maple syrup” odor, starting from about the third day of life. Menkes (1959) reported data collected from six more children. All showed symptoms similar to those described above and died within 15 days to 20 months of birth. In one case, Menkes was able to obtain urine samples during the last months of the infant’s life. When he treated the urine with 2,4-dinitrophenylhydrazone, which forms colored precipitates with keto compounds, he found three α -keto acids in unusually large amounts: a. These α -keto acids are produced by the deamination of amino acids. For each of the α -keto acids above, draw and name the amino acid from which it was derived. Dancis, Levitz, and Westall (1960) collected further data that led them to propose the biochemical defect shown in Figure 18-28. In one case, they examined a patient whose urine first showed the maple syrup odor when he was 4 months old. At the age of 10 months (March 1956), the child was admitted to the hospital because he had a fever, and he showed grossly delayed motor development. At the age of 20 months (January 1957), he was readmitted and was found to have the degenerative neurological symptoms seen in previous cases of maple syrup urine disease; he died soon aer. Results of his blood and urine analyses are shown in the table, along with normal values for each component. Urine concentration (mg/24 h) Plasma concentration (mg/mL) Norma l Patient Norma l Patien t Amino acid(s) Mar. 1 Jan. 1 Jan. 1 Alanin e 5–15 0.2 0.4 3.0–4.8 0.6 Aspara gine and glutam ine 5–15 0.4 0 3.0–5.0 2.0 Asparti c acid 1–2 0.2 1.5 0.1–0.2 0.04 Arginin 1.5–3 0.3 0.7 0.8–1.4 0.8 e Cystin e 2–4 0.5 0.3 1.0–1.5 0 Gluta mic acid 1.5–3 0.7 1.6 1.0–1.5 0.9 Glycin e 20–40 4.6 20.7 1.0–2.0 1.5 Histidi ne 8–15 0.3 4.7 1.0–1.7 0.7 Isoleuc ine 2–5 2.0 13.5 0.8–1.5 2.2 Leucin e 3–8 2.7 39.4 1.7–2.4 14.5 Lysine 2–12 1.6 4.3 1.5–2.7 1.1 Methio nine 2–5 1.4 1.4 0.3–0.6 2.7 Ornithi ne 1–2 0 1.3 0.6–0.8 0.5 Phenyl alanin e 2–4 0.4 2.6 1.0–1.7 0.8 Proline 2–4 0.5 0.3 1.5–3.0 0.9 Serine 5–15 1.2 0 1.3–2.2 0.9 Taurin e 1–10 0.2 18.7 0.9–1.8 0.4 Threon ine 5–10 0.6 0 1.2–1.6 0.3 Trypto phan 3–8 0.9 2.3 Not measu red 0 Tyrosi ne 4–8 0.3 3.7 1.5–2.3 0.7 Valine 2–4 1.6 15.4 2.0–3.0 13.1 b. The table includes taurine, an amino acid not normally found in proteins. Taurine is oen produced as a byproduct of cell damage. Its structure is Based on its structure and the information in this chapter, what is the most likely amino acid precursor of taurine? Explain your reasoning. c. Compared with the normal values given in the table, which amino acids showed significantly elevated levels in the patient’s blood in January 1957? Which ones in the patient’s urine? Based on their results and their knowledge of the pathway shown in Figure 18-28, Dancis and coauthors concluded that “although it appears most likely to the authors that the primary block is in the metabolic degradative pathway of the branched-chain amino acids, this cannot be considered established beyond question.” d. How do the data presented here support this conclusion? e. Which data presented here do not fit this model of maple syrup urine disease? How do you explain these seemingly contradictory data? f. What data would you need to collect to be more secure in your conclusion? References Dancis, J., M. Levitz, and R. Westall. 1960. Maple syrup urine disease: branched-chain ketoaciduria. Pediatrics 25:72–79. Menkes, J.H. 1959. Maple syrup disease: isolation and identification of organic acids in the urine. Pediatrics 23:348– 353. Menkes, J.H., P.L. Hurst, and J.M. Craig. 1954. A new syndrome: progressive familial infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14:462–466.