CHAPTER 19 (Lippincott) Amino Acids - Disposal of Nitrogen OVERALL NITROGEN METABOLISM 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 contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism. This chapter describes how the nitrogen of amino acids is converted to urea and the rare disorders that accompany defects in urea biosynthesis. BIOMEDICAL IMPORTANCE Formation and elimination in the body Ammonia is a metabolic product of amino acid deamination catalyzed by enzymes. Ammonia is quickly converted to urea, which is much less toxic, particularly less basic. This urea is a major component of the dry weight of urine. The liver converts ammonia to urea through a series of reactions known as the urea cycle. Liver dysfunction, such as that seen in cirrhosis, may lead to elevated amounts of ammonia in the blood (hyperammonemia). Likewise, defects in the enzymes responsible for the urea cycle, leads to this disorder. It causes confusion and coma, neurological problems, and aciduria (acid in the urine). While ammonia, derived mainly from the α-amino nitrogen of amino acids, is highly toxic, tissues convert ammonia to the amide nitrogen of nontoxic GLUTAMINE. Subsequent deamination of glutamine in the liver releases ammonia, which is then converted to nontoxic urea. If liver function is compromised, as in cirrhosis or hepatitis, elevated blood ammonia levels generate clinical signs and symptoms. Rare metabolic disorders involve each of the five urea cycle enzymes. PROTEIN TURNOVER OCCURS IN ALL FORMS OF LIFE The continuous degradation and synthesis of cellular proteins occur in all forms of life. Each day humans turn over 1–2% of their total body protein, principally muscle protein. High rates of protein degradation occur in tissues undergoing structural rearrangement—eg, uterine tissue during pregnancy or skeletal muscle in starvation. Of the liberated amino acids, approximately 75% are reutilized. The excess nitrogen forms urea. Since excess amino acids are not stored, those not immediately incorporated into new protein are rapidly degraded. Most proteins in the body are constantly being synthesized and then degraded, permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis 1 determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive, that is, relatively constant, and cellular levels of the protein are controlled by selective degradation. Rate of turnover: In healthy adults, the total amount of protein in the body remains constant, because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300–400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (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, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable, and have half-lives measured in months or years. PROTEASES & PEPTIDASES DEGRADE PROTEINS TO AMINO ACIDS The susceptibility of a protein to degradation is expressed as its half-life (t1/2),, the time required to lower its concentration to half the initial value. Half-lives of liver proteins range from under 30 minutes to over 150 hours. The half-life of a protein is influenced by the nature of the N- terminal residue. For example, proteins that have serine as the N-terminal amino acid are long-lived, with a half-life of more than 20 hours. In contrast, proteins with aspartate as the N-terminal amino acid have a half-life of only 3 minutes. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly degraded and, therefore, exhibit short intracellular half-lives 1. Typical “housekeeping” enzymes have t1/2 values of over 100 hours. 2. Many key regulatory enzymes have a t1/2 of 0.5–2 hours. 3. Degradation of circulating peptides such as hormones follows loss of a sialic acid moiety from the nonreducing ends of their oligosaccharide chains. Asialoglycoproteins are internalized by liver cell asialoglycoprotein receptors and degraded by lysosomal proteases termed cathepsins. 4. Extracellular, membrane-associated, and long-lived intracellular proteins are degraded in lysosomes by ATP-independent processes. 5. Degradation of abnormal and other short-lived proteins occurs in the cytosol and requires ATP and ubiquitin. Ubiquitin, so named because it is present in all eukaryotic cells, is a small (8.5 kDa) protein that targets many intracellular proteins for degradation. Degradation occurs in a multicatalytic complex of proteases known as the proteasome. 2 PROTEIN DEGRADATION: There are two major enzyme systems responsible for degrading damaged or unneeded proteins: 1. The ATP-dependent UBIQUITIN-PROTEASOME SYSTEM of the cytosol, Ubiquitin- proteasome proteolytic pathway: Proteins selected for degradation by the ubiquitin- proteasome system are first covalently attached to ubiquitin, a small, globular, non-enzymic protein. Ubiquitination of the target substrate occurs through linkage of the α-carboxyl group of the C-terminal glycine of ubiquitin to the ε-amino group of a lysine on the protein substrate by a three-step, enzyme-catalyzed, ATP-dependent process. The consecutive addition of ubiquitin moieties generates a polyubiquitin chain. Proteins tagged with ubiquitin are then recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome, which functions like a garbage disposal . 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-proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires energy in the form of ATP. 2.The ATP-independent DEGRADATIVE ENZYME SYSTEM OF THE LYSOSOMES. Proteasomes degrade mainly endogenous proteins, that is, proteins that were synthesized within the cell. Lysosomal enzymes (acid hydrolases, degrade primarily extra-cellular proteins, such as plasma proteins that are taken into the cell by endocytosis, and cell-surface membrane proteins that are used in receptor-mediated endocytosis. These two systems yield AMINO ACIDS for further catabolism. Chemical signals for protein degradation: Because proteins have different half-lives, it is clear that protein degradation cannot 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 are preferentially degraded. The half-life of a protein is influenced by the nature of the N-terminal residue. For example, proteins that have serine as the N-terminal amino acid are long-lived, with a half-life of more than 20 hours. In contrast, proteins with aspartate as the N-terminal amino acid have a half-life of only 3 minutes. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly degraded and, therefore, exhibit short intracellular half-lives. CATABOLISM OF AMINO ACIDS Unlike fats and carbohydrates, amino acids are not stored by the body, that is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from normal protein degradation. Any amino acid in excess of the biosynthetic needs of the cell are rapidly degraded. 3 1. The first phase of catabolism involves the removal of the α-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding α-keto acid—the “carbon skeletons” of amino acids. A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea, which is quantitatively the most important route for disposing off nitrogen from the body. 2. In the second phase of amino acid catabolism, the carbon skeletons are converted to common intermediates of energy producing, metabolic pathways. These compounds can be metabolized to CO2 and water, glucose, fatty acids, or ketone bodies by the central path-ways of metabolism. The role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover. THE AMINO ACID POOL Free amino acids are present throughout the body, for example, in cells, blood, and the extracellular fluids. For the purpose of this discussion, envision all these amino acids as if they belonged to a single entity, called the amino acid pool. This pool is supplied by three sources: 1) amino acids provided by the degradation of body proteins, 2) amino acids derived from dietary protein, 3) synthesis of nonessential amino acids from simple intermediates of metabolism Conversely, the amino pool is depleted by three routes: 1) synthesis of body protein 2) amino acids consumed as precursors of essential nitrogen-containing small molecules, 3) conversion of amino acids to glucose, glycogen, fatty acids, ketone bodies, or CO2 + H2O). Although 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 whole-body nitrogen metabolism TRANSPORT OF AMINO ACIDS INTO CELLS The concentration of free amino acids in the extracellular fluid is significantly lower than that within the cells of the body.