Nitrogen Metabolism PDF

UNIT IV: Nitrogen Metabolism Amino Acids: Disposal of Nitrogen 19...

UNIT IV: Nitrogen Metabolism Amino Acids: Disposal of Nitrogen 19 6-P Gluconate Glycogen Galactose Ribulose 5-P 6-P Gluconolactone UDP-Glucose Galactose 1-P Ribose 5-P Glucose 1-P UDP-Galactose Xylulose 5-P Glucose 6-P Glucose Sedoheptulose 7-P Fructose 6-P Fructose Erythrose 4-P I. OVERVIEW Fructose 1,6-bis-P Glyceraldehyde Fructose 1-P Glyceraldehyde 3-P Dihydroxyacetone-P Glyceraldehyde 3-P 1,3-bis-Phosphoglycerate Glycerol-P Glycerol Unlike fats and carbohydrates, amino acids are not stored by the body, 3-Phosphoglycerate Triacylglycerol that is, no protein exists whose sole function is to maintain a supply of 2-Phosphoglycerate Ala Fatty acyl CoA Fatty acid amino acids for future use. Therefore, amino acids must be obtained Cys Gly Ser Phosphoenolpyruvate Lactate Pyruvate Malonyl CoA from the diet, synthesized de novo, or produced from normal protein Thr Try CO2 CO2 Leu Phe NH3 degradation. Any amino acids in excess of the biosynthetic needs of the CO2 Asn Acetyl-CoA Acetoacetate Tyr Trp Lys Carbamoyl-P cell are rapidly degraded. The first phase of catabolism involves the Citrulline Aspartate Oxaloacetate Citrate β-Hydroxybutyrate removal of the α-amino groups (usually by transamination and subse- Argininosuccinate Malate Isocitrate CO2 Gln Ornithine Pro quent oxidative deamination), forming ammonia and the corresponding Fumarate α-Ketoglutarate CO2 Glu His Arg α-keto acid—the “carbon skeletons” of amino acids. A portion of the Urea Arginine Succinate Succinyl CoA Methylmalonyl CoA Ile Propionyl-CoA free ammonia is excreted in the urine, but most is used in the synthesis Phe Tyr Met Val Acetyl CoA Thr Fatty acyl-CoA of urea (Figure 19.1), which is quantitatively the most important route (odd-number carbons) for disposing of nitrogen from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the NH3 CO2 α-ketoacids are converted to common intermediates of energy produc- Carbamoyl-P ing, metabolic pathways. These compounds can be metabolized to CO2 Aspartate Citrulline and water, glucose, fatty acids, or ketone bodies by the central path- ways of metabolism described in Chapters 8–13, and 16. Argininosuccinate Ornithine II. OVERALL NITROGEN METABOLISM Arginine Urea Amino acid catabolism is part of the larger process of the metabolism of nitrogen-containing molecules. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids con- Figure 19.1 tained in dietary protein. Nitrogen leaves the body as urea, ammonia, Urea cycle shown as part of the essential reactions of energy and other products derived from amino acid metabolism. The role of metabolism. (See Figure 8.2, p. 92, body proteins in these transformations involves two important concepts: for a more detailed view of the the amino acid pool and protein turnover. metabolic pathway.) 245 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 246 246 19. Amino Acids: Disposal of Nitrogen TURNOVER A. Amino acid pool Protein turnover results from the Free amino acids are present throughout the body, for example, in simultaneous synthesis and degradation of protein molecules. cells, blood, and the extracellular fluids. For the purpose of this dis- In healthy, fed adults the total cussion, envision all these amino acids as if they belonged to a amount of protein in the body single entity, called the amino acid pool. This pool is supplied by remains constant because the rate of protein synthesis is just sufficient three sources: 1) amino acids provided by the degradation of body to replace the protein that is proteins, 2) amino acids derived from dietary protein, and 3) degraded. synthesis of nonessential amino acids from simple intermediates of metabolism (Figure 19.2). Conversely, the amino pool is depleted Dietary protein by three routes: 1) synthesis of body protein, 2) amino acids con- Can vary from none (for sumed as precursors of essential nitrogen-containing small example, fasting) to over molecules, and 3) conversion of amino acids to glucose, glycogen, 600 g/day (high protein diets); 100 g/day is typical fatty acids, ketone bodies, or CO2 + H2O (Figure 19.2). Although of the U.S. diet. the amino acid pool is small (comprised of about 90–100 g of amino acids) in comparison with the amount of protein in the body (about 12 kg in a 70-kg man), it is conceptually at the center of Body Synthesis of nonessential whole-body nitrogen metabolism. protein ~400 g/day amino acids In healthy, well-fed individuals, the input to the Varies amino acid pool is balanced by the output, that is, the amount of amino acids contained in the pool is constant. The amino acid pool is said to Amino acid pool be in a steady state, and the individual is said to be in nitrogen balance. ~30 g/day B. Protein turnover Body Synthesis of: protein Porphyrins Most proteins in the body are constantly being synthesized and then ~400 g/day Creatine Neurotransmitters degraded, permitting the removal of abnormal or unneeded pro- Purines teins. For many proteins, regulation of synthesis determines the Pyrimidines concentration of protein in the cell, with protein degradation assum- Other nitrogen- containing ing a minor role. For other proteins, the rate of synthesis is constitu- compounds tive, that is, relatively constant, and cellular levels of the protein are Varies controlled by selective degradation. 1. Rate of turnover: In healthy adults, the total amount of protein in Ketone bodies, the body remains constant, because the rate of protein synthesis Glucose, glycogen fatty acids, is just sufficient to replace the protein that is degraded. This pro- steroids cess, called protein turnover, leads to the hydrolysis and resyn- thesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins 2O (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, con- The amino acids not used in stitute the majority of proteins in the cell. Structural proteins, such biosynthetic reactions are as collagen, are metabolically stable, and have half-lives mea- burned as a fuel. sured in months or years. 2. Protein degradation: There are two major enzyme systems Figure 19.2 responsible for degrading damaged or unneeded proteins: the Sources and fates of amino acids. ATP-dependent ubiquitin-proteasome system of the cytosol, and the ATP-independent degradative enzyme system of the lyso- 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 247 III. Digestion of Dietary Proteins 247 somes. Proteasomes degrade mainly endogenous proteins, that is, proteins that were synthesized within the cell. Lysosomal Protein selected for 1 degradation is tagged enzymes (acid hydrolases, see p. 162) degrade primarily extra- with molecules of cellular proteins, such as plasma proteins that are taken into the ubiquitin. cell by endocytosis, and cell-surface membrane proteins that are Ubiquitinated proteins used in receptor-mediated endocytosis. 2 are recognized by the cytosolic proteasome, which unfolds, de- a. Ubiquitin-proteasome proteolytic pathway: Proteins selected ubiquitinates, and transports the protein for degradation by the ubiquitin-proteasome system are first to its proteolytic core covalently attached to ubiquitin, a small, globular, non-enzymic (an ATP-dependent process). protein. Ubiquitination of the target substrate occurs through linkage of the α-carboxyl group of the C-terminal glycine of Tandemly ubiquitin to the ε-amino group of a lysine on the protein sub- linked molecules strate by a three-step, enzyme-catalyzed, ATP-dependent pro- of ubiquitin cess. The consecutive addition of ubiquitin moieties generates a polyubiquitin chain. Proteins tagged with ubiquitin are then Cellular protein p recognized by a large, barrel-shaped, macromolecular, prote- olytic complex called a proteasome, which functions like a Ubiquitin garbage disposal (Figure 19.3). The proteasome unfolds, deu- biquitinates, and cuts the target protein into fragments that are then further degraded to amino acids, which enter the amino acid pool. [Note: The ubiquitins are recycled.] It is noteworthy that the selective degradation of proteins by the ubiquitin-pro- teosome complex (unlike simple hydrolysis by proteolytic ATP AMP + PPi Proteasome enzymes) requires energy in the form of ATP. b. Chemical signals for protein degradation: Because proteins have different half-lives, it is clear that protein degradation can- Recycled Ubiquitin not be random, but rather is influenced by some structural aspect of the protein. For example, some proteins that have been chemically altered by oxidation or tagged with ubiquitin Non-specific are preferentially degraded. The half-life of a protein is influ- proteases enced by the nature of the N-terminal residue. For example, proteins that have serine as the N-terminal amino acid are Amino acids long-lived, with a half-life of more than 20 hours. In contrast, proteins with aspartate as the N-terminal amino acid have a Peptide fragments produced half-life of only 3 minutes. Additionally, proteins rich in 3 by the proteasome are degraded to amino acids in sequences containing proline, glutamate, serine, and threo- the cytosol. nine (called PEST sequences after the one-letter designations for these amino acids) are rapidly degraded and, therefore, Figure 19.3 exhibit short intracellular half-lives. The ubiquitin-proteasome degradation pathway of proteins. III. DIGESTION OF DIETARY PROTEINS Most of the nitrogen in the diet is consumed in the form of protein, typi- cally amounting to 70–100 g/day in the American diet (see Figure 19.2). Proteins are generally too large to be absorbed by the intestine. [Note: An example of an exception to this rule is that newborns can take up maternal antibodies in breast milk.] They must, therefore, be hydrolyzed to yield di- and tripeptides as well as individual amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine (Figure 19.4). 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 248 248 19. Amino Acids: Disposal of Nitrogen A. Digestion of proteins by gastric secretion The digestion of proteins begins in the stomach, which secretes gastric juice—a unique solution containing hydrochloric acid and the Dietary protein proenzyme, pepsinogen. 1. Hydrochloric acid: Stomach acid is too dilute (pH 2–3) to hydrolyze proteins. The acid, secreted by the parietal cells, func- Pepsin STOMACH tions instead to kill some bacteria and to denature proteins, thus Polypeptides making them more susceptible to subsequent hydrolysis by and amino acids proteases. Trypsin TO LIVER Chymotrypsin Elastase 2. Pepsin: This acid-stable endopeptidase is secreted by the chief PANCREAS Carboxy- cells of the stomach as an inactive zymogen (or proenzyme), peptidase pepsinogen. In general, zymogens contain extra amino acids in Oligopeptides their sequences that prevent them from being catalytically active. and amino acids [Note: Removal of these amino acids permits the proper folding Amino- required for an active enzyme.] Pepsinogen is activated to SMALL peptidases INTESTINE Di- and tri- pepsin , either by HCl, or autocatalytically by other pepsin peptidases molecules that have already been activated. Pepsin releases Amino acids peptides and a few free amino acids from dietary proteins. B. Digestion of proteins by pancreatic enzymes On entering the small intestine, large polypeptides produced in the stomach by the action of pepsin are further cleaved to oligopeptides Figure 19.4 and amino acids by a group of pancreatic proteases. Digestion of dietary proteins by the proteolytic enzymes of the gastro- 1. Specificity: Each of these enzymes has a different specificity for intestinal tract. the amino acid R-groups adjacent to the susceptible peptide bond (Figure 19.5). For example, trypsin cleaves only when the carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthe- sized and secreted as inactive zymogens. 2. Release of zymogens: The release and activation of the pancre- atic zymogens is mediated by the secretion of cholecystokinin and secretin, two polypeptide hormones of the digestive tract (see p. 176). 3. Activation of zymogens: Enteropeptidase (formerly called enter- okinase)—an enzyme synthesized by and present on the luminal surface of intestinal mucosal cells of the brush border mem- brane—converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the N-terminus of trypsinogen. Trypsin subsequently converts other trypsinogen molecules to trypsin by cleaving a limited number of specific peptide bonds in the zymogen. Enteropeptidase thus unleashes a cascade of pro- teolytic activity, because trypsin is the common activator of all the pancreatic zymogens (see Figure 19.5). 4. Abnormalities in protein digestion: In individuals with a defi- ciency in pancreatic secretion (for example, due to chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas), the digestion and absorption of fat and protein are incomplete. This results in the abnormal appearance of lipids (called steatorr- hea, see p. 177) and undigested protein in the feces. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 249 IV. Transport of Amino Acids into Cells 249 SMALL INTESTINE Trp Tyr Ala Ile Phe Ala A Leu B Arg Arg Met Gly or Lys Lys Leu Ser Val R R R R +H N C C NH C C NH C C NH C C NH C C NH C C NH C C NH C C O 3 H O H O H O H O H O H O H O H O Dietary Carboxypeptidase A protein Trypsin Chymotrypsin Elastase Carboxypeptidase B Enteropeptidase Trypsinogen Chymotrypsinogen Proelastase Procarboxypeptidase A Procarboxypeptidase B Figure 19.5 Cleavage of dietary protein by proteases from the pancreas. The peptide bonds susceptible to hydrolysis are shown for each of the five major pancreatic proteases. [Note:The first three are serine endopeptidases, whereas the last two are exopeptidases.] Celiac disease (celiac sprue) is a disease of mal- absorption resulting from immune-mediated damage to the small intestine in response to ingestion of gluten (or gliadin produced from gluten), a protein found in wheat, barley and rye. Proximal convoluted tubule C. Digestion of oligopeptides by enzymes of the small intestine Cystinuria is a disorder of the proximal tubule’s reabsorption of The luminal surface of the intestine contains aminopeptidase—an filtered cystine and dibasic amino exopeptidase that repeatedly cleaves the N-terminal residue from acids (ornithine, arginine, lysine). oligopeptides to produce even smaller peptides and free amino acids. D. Absorption of amino acids and small peptides Free amino acids are taken into the enterocytes by a Na+-linked secondary transport system of the apical membrane. Di- and tri- Cystine Cystine peptides, however, are taken up by a H+-linked transport system. Ornithine Ornithine Arginine Arginine The peptides are hydrolyzed in the cytosol to amino acids that are Lysine Lysine released into the portal system by facilitated diffusion. Thus, only free amino acids are found in the portal vein after a meal containing The inability to reabsorb protein. These amino acids are either metabolized by the liver or cystine leads to accumulation released into the general circulation. [Note: Branched-chain amino and subsequent precipitation of stones of cystine in the acids are important examples of amino acids that are not metabo- urinary tract. lized by the liver, but instead are sent from the liver primarily to mus- cle via the blood.] Figure 19.6 Genetic defect seen in cystinuria. IV. TRANSPORT OF AMINO ACIDS INTO CELLS [Note: Cystinura is distinct from cystinosis, a rare defect in the The concentration of free amino acids in the extracellular fluids is transport of cystine out of lyso- significantly lower than that within the cells of the body. This concentra- somes that results in the formation of cystine crystals within the tion gradient is maintained because active transport systems, driven by lysosome, and tissue damage.] the hydrolysis of ATP, are required for movement of amino acids from 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 250 250 19. Amino Acids: Disposal of Nitrogen the extracellular space into cells. At least seven different transport sys- – COO tems are known that have overlapping specificities for different amino CH2 acids. The small intestine and the proximal tubule of the kidney have R CH2 common transport systems for amino acid uptake; therefore, a defect HC NH3+ O C in any one of these systems results in an inability to absorb particular – COO COO– amino acids into the gut and into the kidney tubules. For example, one α-Amino acid α-Ketoglutarate system is responsible for the uptake of cystine and the dibasic amino acids, ornithine, arginine, and lysine (represented as “COAL”). In the inherited disorder cystinuria, this carrier system is defective, and all Aminotransferase four amino acids appear in the urine (Figure 19.6). Cystinuria occurs at – COO a frequency of 1 in 7,000 individuals, making it one of the most com- R CH2 mon inherited diseases, and the most common genetic error of amino C O CH – + 2 acid transport. The disease expresses itself clinically by the precipita- COO H3N CH tion of cystine to form kidney stones (calculi), which can block the uri- COO– nary tract. Oral hydration is an important part of treatment for this α-Keto acid Glutamate disorder. [Note: Defects in the transport of tryptophan (and other neu- tral amino acids) can result in Hartnup disorder and pellagra-like (see Figure 19.7 p. 380) dermatologic and neurologic symptoms.] Aminotransferase reaction using α-ketoglutarate as the amino- V. REMOVAL OF NITROGEN FROM AMINO ACIDS group acceptor. The presence of the α-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the α-amino group is essen- tial for producing energy from any amino acid, and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted, with the carbon skele- tons being metabolized. This section describes transamination and oxidative deamination—reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 253). A Alanine aminotransferase Alanine α-Ketoglutarate A. Transamination: the funneling of amino groups to glutamate The first step in the catabolism of most amino acids is the transfer of their α-amino group to α-ketoglutarate (Figure 19.7). The products ALT are an α-keto acid (derived from the original amino acid) and gluta- mate. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thus becom- Pyruvate Glutamate ing glutamate. Glutamate produced by transamination can be oxida- tively deaminated (see below), or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino B Aspartate aminotransferase groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (formerly called trans - Oxaloacetate Glutamate aminases). These enzymes are found in the cytosol and mitochon- dria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. All amino acids, with the exception of AST lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their α-amino Aspartate α-Ketoglutarate groups by deamination (see pp. 265–266).] 1. Substrate specificity of aminotransferases: Each aminotrans- ferase is specific for one or, at most, a few amino group donors. Figure 19.8 Aminotransferases are named after the specific amino group Reactions catalyzed during amino acid catabolism. A. Alanine amino- donor, because the acceptor of the amino group is almost transferase (ALT). B. Aspartate always α-ketoglutarate. The two most important aminotrans- aminotransferase (AST). ferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), Figure 19.8). 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 251 V. Removal of Nitrogen from Amino Acids 251 a. Alanine aminotransferase (ALT): ALT is present in many tis- sues. The enzyme catalyzes the transfer of the amino group O O of alanine to α-ketoglutarate, resulting in the formation of C O– C O– + pyruvate and glutamate. The reaction is readily reversible. NH3 C H O C However, during amino acid catabolism, this enzyme (like H C H H C H most aminotransferases) functions in the direction of gluta- H C H H C H – – mate synthesis. Thus, glutamate, in effect, acts as a “collector” C O C O O O of nitrogen from alanine. Glutamate α-Ketoglutarate b. Aspartate aminotransferase (AST): AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST transfers O H C CH2 NH2 amino groups from glutamate to oxaloacetate, forming aspar- P CH2 P CH2 OH OH tate, which is used as a source of nitrogen in the urea cycle (see p. 253). [Note: The AST reaction is also reversible.] CH3 CH3 N N 2. Mechanism of action of aminotransferases: All aminotrans- Pyridoxal Pyridoxamine phosphate phosphate ferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B 6, see p. 378), which is covalently linked to the ε-amino group of a specific lysine residue at the active site of the O O enzyme. Aminotransferases act by transferring the amino group C O– C O– of an amino acid to the pyridoxal part of the coenzyme to gener- + NH3 C H O C ate pyridoxamine phosphate. The pyridoxamine form of the coen- H C H H C H zyme then reacts with an α-keto acid to form an amino acid, at C O– C O– the same time regenerating the original aldehyde form of the O O coenzyme. Figure 19.9 shows these two component reactions for Aspartate Oxaloacetate the reaction catalyzed by AST. 3. Equilibrium of transamination reactions: For most transamina- Figure 19.9 tion reactions, the equilibrium constant is near one. This allows Cyclic interconversion of pyridoxal the reaction to function in both amino acid degradation through phosphate and pyridoxamine removal of α-amino groups (for example, after consumption of a phosphate during the aspartate protein-rich meal) and biosynthesis through addition of amino aminotransferase reaction. groups to the carbon skeletons of α-keto acids (for example, [Note: P = phosphate group.] when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells). Increase above upper normal values 4. Diagnostic value of plasma aminotransferases: Aminotrans- ferases are normally intracellular enzymes, with the low levels Alanine amino- found in the plasma representing the release of cellular contents 20x transferase (ALT ) during normal cell turnover. The presence of elevated plasma levels of aminotransferases indicates damage to cells rich in 15x these enzymes. For example, physical trauma or a disease pro- cess can cause cell lysis, resulting in release of intracellular 10x enzymes into the blood. Two aminotransferases — AST and Bilirubin ALT—are of particular diagnostic value when they are found in 5x the plasma. 0 a. Liver disease: Plasma AST and ALT are elevated in nearly all 0 12 24 36 48 liver diseases, but are particularly high in conditions that Time after ingestion (hours) cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the latter is more sen- Figure 19.10 sitive because the liver contains larger amounts of AST. Pattern of serum alanine amino- transferase (ALT) and bilirubin in Serial enzyme measurements are often useful in determining the plasma, following poisoning the course of liver damage. Figure 19.10 shows the early with the toxic mushroom Amanita release of ALT into the serum, following ingestion of a liver phalloides. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 252 252 19. Amino Acids: Disposal of Nitrogen toxin. [Note: Elevated serum bilirubin results from hepato- NAD+ NADH NH3 cellular damage that decreases the hepatic conjugation and – excretion of bilirubin (see p. 284).] COO COO– CH2 CH2 b. Nonhepatic disease: Aminotransferases may be elevated in Glutamate CH2 dehydrogenase CH2 nonhepatic disease, such as myocardial infarction and mus- + H3N CH O C cle disorders. However, these disorders can usually be distin- COO– COO– guished clinically from liver disease. + NADP NADPH NH3 Glutamate α-Ketoglutarate B. Glutamate dehydrogenase: the oxidative deamination of amino acids Figure 19.11 In contrast to transamination reactions that transfer amino groups, Oxidative deamination by oxidative deamination by glutamate dehydrogenase results in the glutamate dehydrogenase. liberation of the amino group as free ammonia (NH 3 ) (Figure 19.11). These reactions occur primarily in the liver and kidney. They provide α-keto acids that can enter the central pathway of energy metabolism, and ammonia, which is a source of nitrogen in urea synthesis. 1. Glutamate dehydrogenase: As described above, the amino A Disposal of amino acids groups of most amino acids are ultimately funneled to glutamate NH3 by means of transamination with α-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination—a reaction catalyzed by glutamate NH2 α-Ketoglutarate NADH dehydrogenase (see Figure 19.11). Therefore, the sequential (NADPH) of action of transamination (resulting in the collection of amino α-amino acids groups from most amino acids onto α-ketoglutarate to produce TRANSAMINATION OXIDATIVE DEAMINATION glutamate) and the oxidative deamination of that glutamate (regenerating α-ketoglutarate) provide a pathway whereby the Aminotransferase Glutamate dehydrogenase amino groups of most amino acids can be released as ammonia. α-Keto acids a. Coenzymes: Glutamate dehydrogenase is unusual in that it NH2 NAD++ can use either NAD+ or NADP+ as a coenzyme (see Figure of (NADP ) glutamate 19.11). NAD+ is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton (Figure 19.12A), and NADPH is used in reductive amination (the simultaneous gain of ammonia cou- B Synthesis of amino acids pled with the reduction of the carbon skeleton, Figure NH3 19.12B). b. Direction of reactions: The direction of the reaction depends NH2 α-Ketoglutarate NADPH on the relative concentrations of glutamate, α-ketoglutarate, of (NADH) α-amino and ammonia, and the ratio of oxidized to reduced co - acids enzymes. For example, after ingestion of a meal containing TRANSAMINATION REDUCTIVE AMINATION protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation Aminotransferase Glutamate dehydrogenase and the formation of ammonia (see Figure 19.12A). [Note: the α-Keto reaction can also be used to synthesize amino acids from the acids NH2 NADP+ + corresponding α-keto acids (see Figure 19.12B).] of (NAD ) glutamate c. Allosteric regulators: Guanosine triphosphate (GTP) is an allosteric inhibitor of glutamate dehydrogenase, whereas adenosine diphosphate (ADP) is an activator. Thus, when Figure 19.12 energy levels are low in the cell, amino acid degradation by Combined actions of aminotransferase glutamate dehydrogenase is high, facilitating energy produc- and glutamate dehydrogenase reactions. tion from the carbon skeletons derived from amino acids. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 253 VI. Urea Cycle 253 2. D-Amino acid oxidase: D-Amino acids (see p. 5) are found in plants and in the cell walls of microorganisms, but are not used in MOST TISSUES the synthesis of mammalian proteins. D-Amino acids are, how- ever, present in the diet, and are efficiently metabolized by the Glutamate kidney and liver. D-Amino acid oxidase (DAO) is an FAD-depen- ATP + NH3 dent peroxisomal enzyme that catalyzes the oxidative deamina- Glutamine synthetase tion of these amino acid isomers, producing α-keto acids, ADP + Pi ammonia, and hydrogen peroxide. The α-keto acids can enter the general pathways of amino acid metabolism, and be reaminated Glutamine to L-isomers, or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates NMDA-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia.] L-amino acid oxidases are components of several snake venoms. Urea C. Transport of ammonia to the liver H2O Two mechanisms are available in humans for the transport of LIVER Glutaminase ammonia from the peripheral tissues to the liver for its ultimate con- NH3 version to urea. The first, found in most tissues, uses glutamine Glutamate synthetase to combine ammonia (NH3) with glutamate to form glu- dehydrogenase tamine—a nontoxic transport form of ammonia (Figure 19.13). The α-Ketoglutarate Glutamate glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia (see p. 256). The second transport mechanism, used primarily by muscle, involves Alanine Alanine Pyruvate amino- transamination of pyruvate (the end product of aerobic glycolysis) to transferase form alanine (see Figure 19.8). Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. Glucose In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose, which can enter the blood and be used by muscle—a pathway called the glucose-alanine cycle. GLUCOSE– ALANINE CYCLE VI. UREA CYCLE Urea is the major disposal form of amino groups derived from amino acids, and accounts for about 90% of the nitrogen-containing compo- MUSCLE Glucose nents of urine. One nitrogen of the urea molecule is supplied by free ammonia, and the other nitrogen by aspartate. [Note: Glutamate is the Alanine immediate precursor of both ammonia (through oxidative deamination by Alanine amino- transferase Pyruvate glutamate dehydrogenase) and aspartate nitrogen (through transamina- tion of oxaloacetate by AST).] The carbon and oxygen of urea are α-Ketoglutarate Glutamate derived from CO2. Urea is produced by the liver, and then is transported Glutamate in the blood to the kidneys for excretion in the urine. dehydrogenase A. Reactions of the cycle NH3 The first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in Amino acids the cytosol (Figure 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.] 1. Formation of carbamoyl phosphate: Formation of carbamoyl Figure 19.13 phosphate by carbamoyl phosphate synthetase I is driven by Transport of ammonia from peripheral tissues to the liver. cleavage of two molecules of ATP. Ammonia incorporated into car- bamoyl phosphate is provided primarily by the oxidative deamina- 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 254 254 19. Amino Acids: Disposal of Nitrogen Tissues other than liver use enzymes 8 of this pathway to make arginine. H COO– CYTOSOL C NH2 C C NH2+ H2N – OOC H NH H2O C O H2N Malate Fumarate CH2 Urea Ornithine is regenerated CH2 6 and transported into the CH2 mitochondrion. HCNH3+ COO – Arginase MITOCHONDRIAL Arginino- MATRIX succinate L-Arginine NH3+ NH3 + NH3+ COO– lyase CH2 CH2 C N CH CH2 CH2 NH CH2 CH2 CH2 CH2 COO– HCNH3+ HCNH3+ CH2 COO – COO – CH2 L-Ornithine L-Ornithine HCNH3+ – COO NH2 Argininosuccinate CO Ornithine trans- O carbamoylase 4 Citrulline is transported out of the mitochondrion. – O P O O– Carbamoyl phosphate NH2 NH2 C O C O Argininosuccinate synthetase NH NH Pi AMP + CH2 CH2 PPi CH2 CH2 CH2 CH2 3H+ ATP + HCNH3+ HCNH3+ 2ADP COO– COO– + – Pi COO L-Citrulline L-Citrulline +H Carbamoyl 3 N CH phosphate CH2 synthetase I COO– The amino group of aspartate CO2 5 provides one of the L-Aspartate + nitrogen atoms of urea. NH3 Carbon dioxide provides 1 the carbon atom of urea. + 2 ATP A te Oxaloacetate Free ammonia provides 2 one of the nitrogen Glutamate α-Ketoglutarate atoms of urea. The enzyme has an absolute 3 requirement for N-acetyl- Fumarate is hydrated to glutamate, which acts 7 malate, which is oxidized as an allosteric activator. to oxaloacetate, which is transaminated to aspartate. Figure 19.14 Reactions of the urea cycle. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 255 VI. Urea Cycle 255 tion of glutamate by mitochondrial glutamate dehydrogenase (see NADH + NH3 Figure 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. Carbamoyl phos- Amino acids α-Ketoglutarate phate synthetase I requires N-acetylglutamate as a positive allosteric activator (see Figure 19.14). [Note: Carbamoyl phos- phate synthetase II participates in the biosynthesis of pyrimidines α-Keto acids Glutamate NAD+ (see p. 302). It does not require N-acetylglutamate, uses glu- tamine as the nitrogen source, and occurs in the cytosol.] Transamination Oxidative 2. Formation of citrulline: The carbamoyl portion of carbamoyl deamination phosphate is transferred to ornithine by ornithine transcar- bamoylase (OTC) as the high-energy phosphate is released as Glutamate Oxaloacetate Pi. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline are basic amino acids that partici- α-ketoglutarate Aspartate pate in the urea cycle, moving across the inner mitochondrial membrane via a cotransporter. They are not incorporated into cellular proteins because there are no codons for these amino acids (see p. 432).] Ornithine is regenerated with each turn of Urea the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle (see p. 109). CO2 Fumarate Arginine 3. Synthesis of argininosuccinate: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The UREA Ornithine CYCLE α-amino group of aspartate provides the second nitrogen that is Argininosuccinate ultimately incorporated into urea. The formation of argininosucci- Carbamoyl Citrulline phosphate nate is driven by the cleavage of ATP to adenosine monophos- phate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. 4. Cleavage of argininosuccinate: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The argi- nine formed by this reaction serves as the immediate precursor of Figure 19.15 urea. Fumarate produced in the urea cycle is hydrated to malate, Flow of nitrogen from amino acids providing a link with several metabolic pathways. For example, the to urea. Amino groups for urea malate can be transported into the mitochondria via the malate synthesis are collected in the form of ammonia and aspartate. shuttle, reenter the tricarboxylic acid cycle, and get oxidized to oxaloacetate (OAA), which can be used for gluconeogenesis (see p. 120). Alternatively, the OAA can be converted to aspartate via transamination (see Figure 19.8), and can enter the urea cycle (see Figure 19.14). 5. Cleavage of arginine to ornithine and urea: Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in Acetate Glutamate Acetyl CoA the liver. Thus, whereas other tissues, such as the kidney, can synthesize arginine by these reactions, only the liver can cleave arginine and, thereby, synthesize urea. + Arginine Hydrolase Synthase 6. Fate of urea: Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the CoA urine. A portion of the urea diffuses from the blood into the intes- N-Acetylglutamate tine, and is cleaved to CO2 and NH3 by bacterial urease. This ammonia is partly lost in the feces, and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are Figure 19.16 elevated, promoting a greater transfer of urea from blood into the Formation and degradation of N- gut. The intestinal action of urease on this urea becomes a clini- acetylglutamate, an allosteric cally important source of ammonia, contributing to the hyperam- activator of carbamoyl phosphate monemia often seen in these patients. Oral administration of synthetase I. neomycin reduces the number of intestinal bacteria responsible for this NH3 production. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 256 256 19. Amino Acids: Disposal of Nitrogen B. Overall stoichiometry of the urea cycle CO NH2 CH2 Aspartate + NH3 + CO2 + 3 ATP + H2O → CH2 urea + fumarate + 2 ADP + AMP + 2 Pi + PPi HCNH3+ COO– Four high-energy phosphate bonds are consumed in the synthesis of Glutamine each molecule of urea; therefore, the synthesis of urea is irreversible, H20 with a large, negative ΔG (see p. 70). [Note: The ATP is replenished Glutaminase by oxidative phosphorylation.] One nitrogen of the urea molecule is supplied by free NH3, and the other nitrogen by aspartate. Glutamate NH3 is the immediate precursor of both ammonia (through oxidative COO– deamination by glutamate dehydrogenase) and aspartate nitrogen CH2 (through transamination of oxaloacetate by AST). In effect, both nitro- gen atoms of urea arise from glutamate, which, in turn, gathers nitro- CH2 gen from other amino acids (Figure 19.15). HCNH3+ COO– C. Regulation of the urea cycle Glutamate N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase I—the rate-limiting step in the urea cycle (see Figure Figure 19.17 19.14). N-Acetylglutamate is synthesized from acetyl coenzyme A Hydrolysis of glutamine to form and glutamate by N-acetylglutamate synthase (Figure 19.16), in a ammonia. reaction for which arginine is an activator. Therefore, the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both a substrate (glutamate) and the regulator of N-acetylglutamate synthesis. This leads to an increased rate of urea synthesis. VII. METABOLISM OF AMMONIA Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed of primarily by formation of urea in the liver. However, the level of ammonia in the blood must be kept very low, COO– because even slightly elevated concentrations (hyperammonemia) are CH2 toxic to the central nervous system (CNS). There must, therefore, be a CH2 metabolic mechanism by which nitrogen is moved from peripheral tis- HCNH3+ sues to the liver for ultimate disposal as urea, while at the same time COO– maintaining low levels of circulating ammonia. Glutamate ATP + NH3 A. Sources of ammonia Glutamine synthetase Amino acids are quantitatively the most important source of ammonia, ADP + Pi because most Western diets are high in protein and provide excess amino acids, which travel to the liver and undergo transdeamination— CO NH2 the linking of aminotransferase and glutamate dehydrogenase reac- CH2 tions—producing ammonia. However, substantial amounts of CH2 ammonia can be obtained from other sources. HCNH3+ COO– 1. From glutamine: The kidneys generate ammonia from glutamine Glutamine by the actions of renal glutaminase (Figure 19.17) and glutamate dehydrogenase. Most of this ammonia is excreted into the urine as NH4+, which provides an important mechanism for maintain- Figure 19.18 ing the body’s acid-base balance through the excretion of pro- Synthesis of glutamine. tons. Ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase. The intestinal mucosal cells obtain 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 257 VII. Metabolism of Ammonia 257 glutamine either from the blood or from digestion of dietary pro- tein. [Note: Intestinal glutamine metabolism produces citrulline, METABOLISM which travels to the kidney and is used to synthesize arginine.] 2. From bacterial action in the intestine: Ammonia is formed from Glutamate NAD(P)+ α-Keto acids urea by the action of bacterial urease in the lumen of the intes- tine. This ammonia is absorbed from the intestine by way of the portal vein and is almost quantitatively removed by the liver via Glutamate conversion to urea. Aminotransferases dehydrogenase 3. From amines: Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to α-Amino acids ammonia by the action of amine oxidase (see p. 286). α-Ketoglutarate NAD(P)H 4. From purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the rings are released as DIET ammonia (see Figure 22.15 and p. 304). BODY PROTEIN B. Transport of ammonia in the circulation Although ammonia is constantly produced in the tissues, it is pres- Glutamate + ATP ent at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver, and the fact that many tissues, partic- ularly muscle, release amino acid nitrogen in the form of glutamine Glutamine synthetase NH3 or alanine, rather than as free ammonia (see Figure 19.13). ADP + Pi 1. Urea: Formation of urea in the liver is quantitatively the most impor- tant disposal route for ammonia. Urea travels in the blood from the Glutamine liver to the kidneys, where it passes into the glomerular filtrate. Glutaminase 2. Glutamine: This amide of glutamic acid provides a nontoxic stor- age and transport form of ammonia (Figure 19.18). The ATP- requiring formation of glutamine from glutamate and ammonia H2O Glutamate by glutamine synthetase occurs primarily in the muscle and liver, but is also important in the CNS where it is the major mecha- H+ Amide nitrogen nism for the removal of ammonia in the brain. Glutamine is found donated in in plasma at concentrations higher than other amino acids—a biosynthetic NH+4 reactions finding consistent with its transport function. Circulating glu- tamine is removed by the liver and the kidneys and deaminated by glutaminase. In the liver, the NH 3 produced is detoxified Carbamoyl URINE phosphate through conversion to urea, and in the kidney it can be used in synthetase I the excretion of protons. The metabolism of ammonia is summa- Urea cycle rized in Figure 19.19. Urea C. Hyperammonemia The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of serum ammonia are normally low (5–35 μmol/L). However, when liver function is compromised, due Figure 19.19 either to genetic defects of the urea cycle or liver disease, blood lev- Metabolism of ammonia. Urea els can rise above 1,000 μmol/L. Such hyperammonemia is a medi- content in the urine is reported as urinary urea nitrogen or UUN. cal emergency, because ammonia has a direct neurotoxic effect on Urea in blood is reported as BUN the CNS. For example, elevated concentrations of ammonia in the (blood urea nitrogen).The enzymes blood cause the symptoms of ammonia intoxication, which include glutamate dehydrogenase, glutamine tremors, slurring of speech, somnolence, vomiting, cerebral edema, synthetase, and carbamoyl phosphate and blurring of vision. At high concentrations, ammonia can cause synthetase I fix ammonia (NH3) into organic molecules. coma and death. The two major types of hyperammonemia are: 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 258 258 19. Amino Acids: Disposal of Nitrogen 1. Acquired hyperammonemia: Liver disease is a common cause Phenylbutyrate is a prodrug that is of hyperammonemia in adults, and may be due, for example, to rapidly converted to phenylacetate, which combines with glutamine to viral hepatitis or to hepatotoxins such as alcohol. Cirrhosis of the form phenylacetylglutamine. The liver may result in formation of collateral circulation around the phenylacetyglutamine, containing two liver. As a result, portal blood is shunted directly into the sys- atoms of nitrogen, is excreted in the urine, thus assisting in clearance of temic circulation and does not have access to the liver. The con- nitrogenous waste. version of ammonia to urea is, therefore, severely impaired, leading to elevated levels of ammonia. URINE 2. Congenital hyperammonemia: Genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1:25,000 live births. Ornithine Phenylacetylglutamine transcarbamoylase deficiency, which is X-linked, is the most com- Protein mon of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal recessive inheritance pat- Phenylacetate tern. In each case, the failure to synthesize urea leads to hyper- Amino acids ammonemia during the first weeks following birth. [Note: The Glutamine hyperammonemia seen with arginase deficiency is less severe Glutamine Glutamine synthetase because arginine contains two waste nitrogens and can be Glutamine Glutamate excreted in the urine.] Historically, urea cycle defects had high Glutamine morbidity (neurological manifestations) and mortality. Treatment NH3 NH3 NH3 included restriction of dietary protein in the presence of sufficient NH3 calories to prevent catabolism. Administration of compounds that NH3 bind covalently to amino acids, producing nitrogen-containing molecules that are excreted in the urine, has improved survival. Figure 19.20 For example, phenylbutyrate given orally is converted to phenylac- Treatment of patients with urea etate. This condenses with glutamine to form phenyl - cycle defects by administration of acetylglutamine, which is excreted (Figure 19.20). phenylbutyrate to aid in excretion of ammonia. VIII. CHAPTER SUMMARY Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids con- tained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (Figure 19.21). Free amino acids in the body are produced by hydrolysis of dietary protein by proteases in the stomach and intestine, degradation of tissue proteins, and de novo synthesis. This amino acid pool is consumed in the synthesis of body protein, metabolized for energy, or its members serve as precursors for other nitrogen-containing compounds. Note that body protein is simultaneously degraded and resynthesized— a process known as protein turnover. For many proteins, regulation of synthesis determines the concentration of the protein in the cell, whereas the amounts of other proteins are controlled by selective degradation. The ATP-dependent ubiquitin/proteasome and ATP-independent lysosomal acid hydrolases are the two major enzyme systems that are responsible for degrading damaged or unneeded proteins. Nitrogen cannot be stored, and amino acids in excess of the biosynthetic needs of the cell are immediately degraded. The first phase of catabolism involves the transfer of the α-amino groups by PLP-dependent transamination, followed by oxidative deamination of glutamate, forming ammonia and the corresponding α-keto acids. A portion of the free ammonia is excreted in the urine, some is used in converting glutamate to glutamine, but most is used in the synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. The two major causes of hyperammonemia (with its CNS effects) are liver disease and inherited deficiencies of enzymes (such as ornithine transcarbamolyase) in the urea cycle. 168397_P245-260.qxd7.0:19 Disposal of nitro 10-11-05 2010.4.4 5:46 PM Page 259 VIII. Chapter Summary 259 Amino acid pool Removal of nitrogen from amino acids is defined as occurs because All the free amino acids in cells and extracelluar fluids Amino acids cannot directly participate in energy metabolism are produced by are consumed by therefore Degradation Synthesis of Degradation Amino groups are removed