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amino acids
i. overview
proteins are the most abundant and functionally diverse molecules in living systems. virtually every life process depends on this class of molecules. for example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. in bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. in the bloodstream, proteins, such as hemoglobin and plasma albumin, shuttle molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. in short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids.
ii. structure of the amino acids
although more than 300 different amino acids have been described in nature, only twenty are commonly found as constituents of mammalian proteins. each amino acid (except for proline, has a carboxyl group, an amino group, and a distinctive side chain ("r-group") bonded to the ?-carbon atom (figure 1.1 a).
at physiologic ph (approximately ph = 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (-coo¯), and the amino group is protonated (-nh3+).in proteins, almost all of these carboxyl and amino groups are combined in peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (figure 1.1b). thus, it is the nature of the side chains that ultimately dictates the role an amino acid plays in a protein.
it is, therefore, useful to classify the amino acids according to the properties of their side chain-that is, whether they are non polar (that is, have an even distribution of electrons) or polar (that is, have an uneven distribution of electrons, such as acids and bases figures 1.2 and 1.3).
amino acids with non polar side chains
each of these amino acids has a non polar side chain that does not bind or give off protons or participate in hydrogen or ionic bonds (see figure 1.2)
the side chains of these amino acids can be thought of as "oily" or lipid like a property that promotes hydropinginginghobic interactions .
1. location of non polar amino acids in proteins:
in proteins found in aqueous solutions, the side chains of the non polar amino acids tend to cluster together in the interior of the protein (figure 1.4). this phenomenon is the result of the hydropinginginghobicity of the non polar r-group which act much like dropinginginglets of oil that coalesce in an aqueous environment. the non polar r-groups thus fill up the interior of the folded protein and help give it its three-dimensional shape. [note: in proteins that are located in a hydropinginginghobic environment, such as a membrane, the non polar r-groups are found on the outside surface of the protein, interacting with the lipid environment (see figure 1.4).] the importance of these hydropinginginghobic interactions is in stabilizing protein structure .
2. proline: the side chain of proline and its ?-amino group form a ring structure, and thus proline differs from other amino acids in that it contains an imino group, rather than an amino group (figure 1.5)
the unique geometry of proline contributes to the formation of the fibrous structure of collagen, and often interrupts the ?-helices found in globular proteins.
b. amino acids with uncharged polar side chains
these amino acids have zero net charge at neutral ph although the side chains of cysteine and tyrosine can lose a proton at an alkaline ph (see figure 1.3).
serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (figure 1.6).
the side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds.
1. disulfide bond: the side chain of cysteine contains a sulfhydryl group (-sh), which is an important component of the active site of many enzymes. in proteins, the -sh groups of two cysteines can become oxidized to form a dimer cystine, which contains a covalent cross-link called a disulfide bond (-s-s-).
2. side chains as sites of attachment for other compounds: serine, threonine, and, rarely, tyrosine contain a polar hydroxyl group that can serve as a site of attachment for structures such as a phosphate group. [note: the side chain of serine is an important component of the active site of many enzymes.] in addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins.
c. amino acids with acidic side chains
the amino acids aspartic and glutamic acid are proton donors. at neutral ph the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (-c00¯). they are, therefore, called aspartate or glutamate to emphasize that these amino acids are negatively charged at physiologic ph (see figure 1.3)
d. amino acids with basic side chains
the side chains of the basic amino acids accept protons (see figure 1.3) at physiologic ph the side chains of lysine and arginine are fully ionized and positively charged. in contrast, histidine is weakly basic, and the free amino acid is largely uncharged at physiologic ph .however, when histidine is incorporated into a protein, its side chain can be either positively charged or neutral, depending on the ionic environment provided by the polypeptide chains of the protein. [note:this is an important property of histidine that contributes to the role it plays in the functioning of proteins such as hemoglobin].
e. abbreviations and symbols for the commonly occurring amino acids
each amino acid name has an associated three-letter abbreviation and a one-letter symbol (figure 1.7). the one-letter codes are determined by the following rules:
1. unique first letter: if only one amino acid begins with a particular letter, then that letter is used as its symbol. for example,i = isoleucine.
2. most commonly occurring amino acids have priority: if more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. for example, glycine is more common than glutamate, so g = glycine.
3. similar sounding names: some one-letter symbols sound like the amino acid they represent. for example, f = phenylalanine, or w= tryptophan ("twyptophan" as elmer fudd would say).
4. letter close to initial letter: for the remaining amino acids, a one-letter symbol is assigned that is as close in the alphabet as possible to the initial of the amino acid. further, b is assigned to asx, signifying either aspartic acid or asparagine, z is assigned to glx signifying either glutamic acid or glutamine and x is assigned to an unidentified amino acid.
f. optical properties of amino acids
the ?-carbon of each amino acid is attached to four different chemical groups and is, therefore, a chiral or optically active carbon atom. glycine is the exception because its ?-carbon has two hydrogen substituents and, therefore, is optically inactive. [note: amino acids that have an asymmetric center at the ?-carbon can exist intwo forms, designated d and l, that are mirror images of each other(figure 1.8)
the two forms in each pair are termed stereoisomers,optical isomers, or enantiomers .all amino acids found in proteins are of the l-configuration. however, d-amino acids are found in some antibiotics and in bacterial cell walls.
amino acids metabolism:
disposal of nitrogen
i. overview
unlike fats and carbohydrates, amino acids are not stored by the body, that is, no proteins exist whose sole function it 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 acids in excess of the biosynthetic needs of the cell are rapidly degraded. the first phase of catabolism involves the removal of the ?-amino groups (usually by transamination and subsequent oxidative deamination, forming ammonia and the corresponding ?-ketoacid-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 of nitrogen from the body.
in the second phase of amino acid catabolism, the carbon skeletons of the ?-ketoacid 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 pathways of metabolism
ii. overall nitrogen metabolism
amino acid catabolism is part of the larger process of whole body nitrogen metabolism. 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. the role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover.
a. amino acid pool
amino acids released by hydrolysis of dietary or tissue protein, or synthesized denovo .mix with other free amino acids distributed throughout the body. collectively, they constitute the amino acid pool (figure 19.2). the amino acid pool, containing about 100 g of amino acids, is small in comparison with the amount of protein in the body (about 12 kg in a 70 kg man).
in the only fate of the amino acid pool were to be used to resynthesize body proteins, adults would not have a significant need for additional dietary protein. however, only about 75 percent of the amino acids obtained through hydrolysis of body protein are recaptured through the biosynthesis of new tissue protein. the remainder are metabolized or serve as precursors for the compounds shown in figure.
in well-fed individuals, this metabolic loss of amino acids is compensated for by dietary protein, which contributes to the amino pool.
b-protein turnover
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 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.
1. 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 to 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.
2. protein degradation: there are two major enzyme systems responsible for degrading damaged or unneeded proteins: the energy-dependent ubiquitin-proteasome mechanism, and the non-energy-dependent degradative enzymes of the lysosomes. proteasomes mainly degrade endogenous proteins, that is, proteins that were synthesized within the cell. lysosomes (see p.
160) primarily degrade extracellular 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.
a-ubiquitin-proteasome proteolytic pathway: proteins destined for degradation by the ubiquitin-proteasome mechanism are first covalently attached to ubiquitin, a small, globular protein. ubiquitination of the target substrate occurs through linkage the ?-carboxyl glycine of ubiquitin to a lysine ?-amino group on the protein substrate. the consecutive addition of ubiquitin moieties generates a polyubiquitin chain. proteins tagged with ubiquitin are then recognized by a large, barrel-shaped,proteolytic molecule called a proteasome which functions like a garbage disposal (figure 19.3). the proteosome cuts the target protein into fragments that are then further degraded to amino acids, which enter the amino acid pool.it is noteworthy that the selective degradation of proteins by the ubiquitin proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires atp, that is, it is energy-dependent.
b. 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 n-terminal residue. 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 residue. for example, proteins that have serine as the n-terminal amino acid are long-lived, with a half-life of more than twenty hours. in contrast, proteins with aspartate as the n-terminal amino acid have a half-life of only three minutes. further, 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.
digestion of dietary proteins
most of the nitrogen in the diet is consumed in the form of protein, typically amounting from 70 to 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 their constituent 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).
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 proenzyme, pepsinogen:
1. hydrochloric acid: stomach acid is too dilute (ph 2 to 3) to hydrolyze proteins. the acid functions instead to kill some bacteria and to denature proteins, thus making them more susceptible to subsequent hydrolysis by proteases.
2. pepsin: this acid-stable endopeptidase is secreted by the serous cells of the stomach as an inactive zymogen (or proenzyme), pepsinogen. in general, zymogens contain extra amino acids in their sequences, which prevent them from being catalytically active. [note: removal of these amino acids permits the proper folding required for an active enzyme.] pepsinogen is activated to pepsin, either by hcl or autocatalytically by other pepsin molecules that have already been activated. pepsin releases 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 and amino acids by a group of pancreatic proteases.
1. specificity: each of these enzymes has a different specificity for 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 synthesized and secreted as inactive zymogens.
2. release of zymogens: the release and activation of the pancreatic zymogens is mediated by the secretion of cholecystokinin and secretin, two polypeptide hormones of the digestive tract.
3. activation of zymogens: enteropeptidase (formerly called enterokinase-an enzyme synthesized by and present on the luminal surface of intestinal mucosal cells of the brush border
membrane-converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the nh2-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 proteolytic 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 deficiency 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 is incomplete. this results in the abnormal appearance of lipids (steatorrhea) and undigested protein in the feces.
c.digestion of oligopeptides by enzymes of the small intestine
the luminal surface of the intestine contains aminopeptidase an- exopeptidase that repeatedly cleaves the n-terminal residue from oligopeptides to produce free amino acids and smaller peptides.
d. absorption of amino acids and dipeptides
free amino acids and dipeptides are taken up by the intestinal epithelial cells. there, the dipeptides are hydrolyzed in the cytosol to amino acids before being released into the portal system. thus, only free amino acids are found in the portal vein after a meal containing protein. these amino acids are either metabolized by the liver or released into the general circulation
iv. transport of amino acids into cells
the concentration of free amino acids in the extracellular fluids is significantly lower than that within the cells of the body. this concentration gradient is maintained because active transport systems, driven by the hydrolysis of atp, are required for movement of amino acids from the extracellular space into cells. at least seven different transport systems are known that have overlapping specificities for different amino acids. for example, one transport system is responsible for reabsorption of the amino acids cystine, ornithine, arginine, and lysine in kidney tubules.in the inherited disorder cystinuria, this carrier system is defective, resulting in the appearance of all four amino acids in the urine (figure 19.6). cystinuria occurs at a frequency of 1 in 7000 individuals, making it one of the most common inherited diseases, and the most common genetic error of amino acid transport. the disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi), which can block the urinary tract. oral hydration is an important part of treatment for this disorder.
v. removal of nitrogen from amino acids
the presence of the ?-amino group keeps amino acids safely locked away from oxidative breakdown. removing the ?-amino group is essential 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 skeletons being metabolized. this section describes transamination and oxidative deamination—reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 251).
a. transamination: the tunneling of amino groups to glutamate
the first step in the catabolism of most amino acids is the transfer of their ?-amino group to ?-ketoglutarate (figure 19.7). the products are an ?-keto acid (derived from the original amino acid) and glutamate. ?-ketoglutarate plays a unique role in amino acid metabolism by accepting the amino groups from other amino acids, thus becoming glutamate. glutamate produced by transamination can be oxidatively deaminated (see below), or used as an amino group donor in the synthesis of nonessential amino acids. this transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (formerly called transaminases). these enzymes are found in the cytosol of cells throughout the body-especially those of the liver, kidney, intestine, and muscle. all amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [note: these two amino acids lose their ?-amino groups by deamination (see p. 264).]
1. substrate specificity of aminotransferases: each aminotransferase is specific for one or, at most, a few amino group donors.
aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always ?-ketoglutarate. the two most important aminotransferase reactions are catalyzed by alanine aminotransferase and aspartate aminotransferase (figure 19.8)
a. alanine aminotransferase (alt), formerly called glutamate pyruvate transaminase(gpt) ,is present in many tissues. the enzyme catalyzes the transfer of the amino group of alanine to ?-ketoglutarate, resulting in the formation of pyruvate and glutamate. the reaction is readily reversible. however, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. thus, glutamate, in effect, acts as a "collector" of nitrogen from alanine.
b. aspartate aminotransferase (ast), formerly called glutamate:oxaloacetate transaminase (got), is an exception of the rule that aminotransferases funnel amino groups to form f glutamate. during amino acid catabolism, ast transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle (see p.251).
2. mechanism of action of aminotransferases: all aminotransferases require the coenzyme pyridoxal phosphate (a derivative) of vitamin b6 ,see p. 376), which is covalently linked to the e-amino group of a specific lysine residue at the active site of the enzyme. aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. the pyridoxamine form of the coenzyme then reacts with an ?-keto acid to form an amino acid at the same time regenerating the original aldehyde form of the coenzyme. figure 19.9 shows these two component reaction of the reaction catalyzed by aspartate aminotransferase.
3. equilibrium of transamination reactions: for most transamination reactions, the equilibrium constant is near one, allowing thereaction to function in both amino acid degradation throughremoval of ?-amino groups (for example, after consumption of aprotein-rich meal), and biosynthesis through addition of aminogroups to the carbon skeletons of ?-keto acids (for example,when the supply of amino acids from the diet is not adequate tomeet the synthetic needs of cells).
4. diagnostic value of plasma aminotransferases: aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contentsduring normal cell turnover. the presence of elevated plasmalevels of aminotransferases indicates damage to cells rich inthese enzymes. for example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellularenzymes into the blood. two aminotransferases ast and alt are of particular diagnostic value when they are found in the plasma.
a. liver disease: plasma ast and alt are elevated in nearly all liver diseases, but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis,toxic injury, and prolonged circulatory collapse. alt is morespecific for liver disease than ast, but the latter is more sensitive because the liver contains larger amounts of ast.serial enzyme measurements are often useful in determiningthe course of liver damage. figure 19.10 shows the earlyrelease of alt into the serum, following ingestion of a livertoxin. [note: elevated serum bilirubin results from heptocellular damage that decreases the hepatic conjugation andexcretion of bilirubin (see p. 282).]
b. nonhepatic disease: aminotransferases may be elevated innonhepatic disease, such as myocardial infarction and muscle disorders. however, these disorders can usually be distinguished clinically from liver disease.
b.glutamate dehydrogenase: the oxidative deamination of amino acids
in contrast to transamination reactions that transfer amino groups,oxidative deamination by glutamate dehydrogenase results in the liberation of the amino group as free ammonia (figure 9.11).these reactions occur primarily in the liver and kidney. they provide?-ketoacids 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 groups of most amino acids are ultimately funneled to glutamate by means of transamination with ?-ketoglutarate. glutamate isunique in that it is the only amino acid that undergoes rapid oxidative deamination-a reaction catalyzed by glutamatedehydrogenase (see figure 19.10). therefore, the sequential action of transamination (resulting in the collection of aminogroups from other amino acids onto ?-ketoglutarate to produce glutamate) and the subsequent oxidative of that glutamate (regenerating ?-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.
a. coenzymes: glutamate dehydrogenase is unusual in that it can use either nad+ or nadp+ as a coenzyme.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 coupled with the reduction of the carbon skeleton, figure 19.12b).
b. direction of reactions: the direction of the reaction depends on the relative concentrations of glutamate,?-ketoglutarate, and ammonia, and the ratio of oxidized to reduced coenzymes. for example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see figure 19.11a).
[note: the reaction can also be used to synthesize amino acids from the corresponding ?-ketoacids (see figure 19.11b)]
c.allosteric regulators: atp and gtp are allosteric inhibitors of glutamate dehydrogenase, whereas adp and gdp are activators of the enzyme. thus, when energy levels are low in the cell, amino acid degradation by glutamate dehydrogenase is high, facilitating energy production from the carbon skeletons derived from amino acids.
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 the synthesis of mammalian proteins. d-amino acids are, however, present in the diet, and are efficiently metabolized by the liver. d-amino acid oxidase is an fad-dependent enzyme that catalyzes the oxidative deamination of these amino acid isomers. the resulting ?-ketoacids can enter the general pathways of amino acid metabolism, and be reaminated to l-isomers, or catabalized for energy.
c. transport of ammonia to the liver
two mechanisms are available in humans for the transport of ammonia from the peripheral tissues to the liver for its ultimate conversion to urea. the first, found in most tissues, uses glutamine synthetase to combine ammonia with glutamate to form glutamine-a non toxic transport form of ammonia (figure 19.13). the glutamine is transported in the blood to the liver where is cleaved by glutaminase to produce glutamate and free ammonia.the second transport mechanism, used primarily by muscle, involves transamination of pyruvate (the end-product of aerobic glyclosysis) to form alanine (see figure 19.8). alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination.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 path way called glucose-alanine cycle.
vi. urea cycle
urea is the major disposal form of amino groups derived from amino acids, and accounts for about ninety percent of the nitrogen-containing components of urine. one nitrogen of the urea molecule is supplied by free nh3 and the other nitrogen by aspartate. [note: glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by aspartate aminotransferase).] the carbon and oxygen of urea are derived from co2. urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine.
a. reactions of the cycle
the first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in the cytosol (figure 19.14).
1. formation of carbamoyl phosphate: formation of carbamoyl phosphate by carbamoyl phosphate synthetase1 is driven by cleavage of two molecules of atp. ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial glutamate dehydrogenase (see figure 19.11) ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea.
carbamoyl phosphate synthetase 1 requires n-acetylglutamate as a positive allosteric activator (see figure 19.14). [note: carbamoyl phosphate synthetase ii participates in the biosynthesis of pyrimidine. it does not require n-acetyl-glutamate, and occurs in the cytosol.]
2. formation of citrulline: ornithine and citrulline are basic amino acids that participate in the urea cycle. [note: they are not incorporated into cellular proteins, because there are no codons for these amino acids (see p. 429).] ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle .the release of the high-energy phosphate of carbamoyl phosphate as inorganic phosphate drives the reaction in the forward direction. the reaction product, citrulline, is transported to the cytosol.
3. synthesis of argininosuccinate: citrulline condenses with aspartate to form argininosuccinate. the ?-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. the formation of argininosuccinate is driven by the cleavage of atp to amp and pyrophosphate(ppi). this is the third and final molecule of atp consumed in the formation of urea.
4. cleavage of argininosuccinate: argininosuccinate is cleaved to yield arginine and fumarate. the arginine formed by this reaction serves as the immediate precursor of urea. fumarate produced in the urea cycle is hydrated to malate, providing a link with several metabolic pathways. for example, the malate can be transported into the mitochondria via the malate shuttle and re-enter the tca cycle. alternatively, cytosolic malate can be oxidized to oxaloacetate, which can be converted to aspartate (see figure19.8) or glucose .
5. cleavage of arginine to ornithine and urea: arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in 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.
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 urine. a portion of the urea diffuses from the blood into the intestine, and is cleaved to nh3 and co2 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 elevated, promoting a greater transfer of urea from blood into the gut. the intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. oral administration of neomycin reduces the number of intestinal bacteria responsible for this nh3 production.
b.overall stoichiometry of the urea cycle
aspartate + nh3 +co2 +3atp? urea +fumarate + 2 adp + amp + 2pi +ppi+ 3 h2o
four high-energy phosphates are consumed in the synthesis of each molecule of urea: two atp are needed to restore two adp to two atp, plus two to restore amp to atp. therefore, the synthesis of urea is irreversible, with a large, negative ag (see p. 70). one nitrogen of the urea molecule is supplied by free nh3,and the other nitrogen by aspartate.glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by aspartate aminotransferase). in effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitrogen from other amino acids (figure 19.15).
c.regulation of the urea cycle
n-acetylglutamate is an essential activator for carbamoyl phosphate synthetase, 1
the- rate-limiting step in the urea cycle (see figure19.14). n-acetylglutamate is synthesized from acetyl coa and glutamate (figure 19.16), in a 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 the 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, because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system (cns). there must, therefore, be a metabolic mechanism by which nitrogen is moved from peripheral tissues to the liver for ultimate disposal as urea, while at the same time low levels of circulating ammonia must be maintained.
a. sources of ammonia
amino acids are quantitatively the most important source of ammonia, because most western diets are high in protein and provide excess amino acids, which are deaminated to produce ammonia. however, substantial amounts of ammonia can be obtained from other sources.
1. from amino acids: many tissues, but particularly the liver, form ammonia from amino acids by the aminotransferase and glutamate dehydrogenase reactions previously described.
2. from glutamine the kidneys form ammonia from glutamine by the action of renal glutaminase (figure). most of this ammonia is excreted into the urine as nh4, which provides an important mechanism for maintaining the body s acid-base balance. ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase. the intestinal mucosal cells obtain glutamine either from the blood or from digestion of dietary protein.
3. from bacterial action in the intestine: ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. this ammonia is absorbed from the intestine by way of the portal vein and is almost quantitatively removed by the liver via conversion to urea.
4. from amines: amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidase .
5. from purines and pyrimidines: in the catabolism of purines and pyrimidines, amino groups attached to the rings are released as ammonia.
b. transport of ammonia in the circulation
although ammonia is constantly produced in the tissues, it is present 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, particularly muscle, release amino acid nitrogen in the form of glutamine or alanine, rather than as free ammonia.
1. urea: formation of urea in the liver is quantitatively the most important disposal route for ammonia. urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate.
2. glutamine:this amide of glutamic acid provides a nontoxic storage and transport form of ammonia (figure). theatp-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in the muscle and liver, but is also important in the nervous system, where it is the major mechanism for the removal of ammonia in the brain. glutamine is found in plasma at concentrations higher than other aminoacids-a finding consistent with its transport function. circulating glutamine is removed by the kidneys and deaminated by glutaminase
the metabolism of ammonia is summarized in figure .
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 to 50 ?mol/l).
however, when the liver function is compromised, due either to genetic defects of the urea cycle, or liver disease, blood levels can rise above 1000 ?mol/l.
such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on cns. for example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence, vomiting, cerebral edema, and blurring of vision. at high concentrations, ammonia can cause coma and death. the two major types of hyperammonemia are:
1. acquired hyperammonemia: liver disease is a common cause of hyperammonemia in adults. it may be a result of an acute process, for example, viral hepatitis, ischemia, or hepatotoxins. cirrhosis of the liver caused by alcoholism, hepatitis, or biliary obstruction may result in formation of collateral circulation around the liver. as a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. the detoxification of ammonia (that is, its conversion to urea) is, therefore, severely impaired, leading to elevated levels of circulating ammonia.
2. hereditary hyperammonemia: genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1 in 30,000 live births, ornithine transcarbamoylase deficiency, which is x-linked, is the most common of these disorders, affecting males predominantly, although female carriers have been clinically affected. all of the other urea cycle disorders follow an autosomal recessive inheritence pattern. in each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. all inherited deficiencies of the urea cycle enzymes result in mental retardation. treatment includes limiting protein in the diet, and administering compounds that bind covalently to amino acids, producing nitrogen-containing molecules that are excreted in the urine. for example, phenylbutyrate given orally is converted to phenylacetate. this condenses with glutamine to form phenylacetylglutamine, which is excreted .
disorder enzyme deficiency
hyperammonaemia type 1 carbamoyl phosphate synthase
hyperammonaemia type 2 ornithine trancarbamoylase
citrullinaemia argininosuccinate synthase
argininosuccinic aciduria argininosuccinase
hyperargininaemia arginase
amino acid degradation and synthesis
i. overview
the catabolism of the amino acids found in proteins involves the removal of ?-amino groups, followed by the breakdown of the resulting carbon skeletons. these pathways converge to form seven intermediate product: oxaloacetate, ?-ketoglutarate, pyruvate,fumarate, succinyl coa, acetyl coa, and acetoacetyl coa. these products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose or lipid, or in the production of energy through their oxidation to co2 and water by the citric acid cycle.nonessential amino acids (figure) can be synthesized in sufficient amounts from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from essential amino acids. in contrast, the essential amino acids cannot be synthesized (or produced in sufficient amounts) by the body and, therefore, must be obtained from the diet in order for normal protein synthesis to occur. genetic defects in the pathways of amino acid metabolism can cause serious disease.
ii.glucogenic and ketogenic amino acids
amino acids can be classified as glucogenic or ketogenic based on which of the seven intermediates are produced during their catabolism (see figure ).
a. glucogenic amino acids
amino acids whose catabolism yields pyruvate or one of the intermediates of the citric acid cycle are termed glucogenic or glycogenic. these intermediates are substrates for gluconeogenesis and, therefore, can give rise to the net formation of glucose or glycogen in the liver and glycogen in the muscle.
b. ketogenic amino acids
amino acids whose catabolism yields either acetoacetate or its precursor, (acetyl coa or acetoacetyl coa) are termed ketogenic (see figure). acetoacetate is one of the ketone bodies which also include 3-hydroxybutyrate and acetone. leucine and lysine are the only exclusively ketogenic amino acids found in proteins. their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net formation of glucose or glycogen in the liver, or glycogen in the muscle.
iii. catabolism of the carbon skeletons of amino acids
the pathways by which amino acids are catabolized are conveniently organized according to which one (or more) of the seven intermediates listed above is produced from a particular amino acid.
a. amino acids that form oxaloacetate
asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate (figure).
b. amino acids that form ?-ketoglutarate
1.glutamine is converted to glutamate and ammonia by the enzyme glutaminase.glutamate is converted to ?-ketoglutarate by transamination, or through oxidative deamination by glutamate dehydrogenase.
2.proline is oxidized to glutamate. glutamate is transaminated or oxidatively deaminated to form ?-ketoglutarate .
3.arginine is cleaved by arginase to produce ornithine. [note: this reaction occurs primarily in the liver as part of the urea cycle .ornithine is subsequently converted to ?-ketoglutarate.
4. histidine is oxidatively deaminated by histidase to urocanic acid, which subsequently forms n-formiminoglutamate (figlu, figure). figlu donates its formimino group to tetrahydrofolate, leaving glutamate, which is degraded as described above.
figure :degradation of histidine
c. amino acids that form pyruvate
1. alanine loses its amino group by transamination to form pyruvate (figure).
2. serine can be converted to glycine and n5,n10-methylene tetrahydrofolate (figure a) serine can also be converted to pyruvate by serine dehydratase (figure b)
3. glycine can either be converted to serine by addition of a methylene group from n5,n10-methylene tetrahydrofolic acid (see figurea), or oxidized to nh4+ and co2.
4. cystine is reduced to cysteine, using nadh+h+ as a reductant. cysteine undergoes desulfuration to yield pyruvate.
5. threonine converted to pyruvate or to ?-ketobutyrate, which forms succinyl coa.
d. amino acids that form fumarate
phenylalanine and tyrosine: hydroxylation of phenylalanine leads to the formation of tyrosine (figure). this reaction, catalyzed by is the first reaction in the catabolism of phenylalanine. thus, the metabolism of phenylalanine and tyrosine merge, leading ultimately to the formation of fumarate and acetoacetate. phenylalanine and tyrosine are, therefore, both glucogenic and ketogenic.
e. amino acids that form succinyl coa: methionine
methionine is one of four amino acids that form succinyl coa. this sulfur-containing amino acid deserves special attention because converted to s-adenosylmethionine (sam), the major methyl-group donor one-carbon metabolism (figure). methionine also the source of homocysteine—a metabolite associated with atherosclerotic vascular disease.
1. synthesis of sam: methionine condenses with atp, forming sam-a high-energy compound that is unusual in that it contains no phosphate. the formation of sam is driven ,in effect, by hydrolysis of al three phosphate bonds in atp (see figure ).
2. activated methyl group: the methyl group attached to the tertiary sulfur in sams "activated," and can be transferred to a variety of acceptor molecules, such as ethanolamine in the synthesis of choline. the methyl groups usually transferred to oxygen or nitrogen atoms, but sometimes to carbon atoms. the reaction product, s-adenosylhomocysteine, is a simple thioether, analogous to methionine. the resulting loss of free energy accompanying the reaction makes methyl transfer essentially irreversible.
3. hydrolysis of sam: after donation of the methyl group, s-adenosylhomocysteine is hydrolyzed to homocysteine and adenosine. homocysteine has two fates. if there is a deficiency of methionine, homocysteine may be remethylated to methionine (see figure).if methionine stores are adequate, homocysteine may enter the trans- sulfuration pathway, where it is converted to cysteine.
a. resynthesis of methionine: homocysteine accepts a methyl group from n5-methyltetrahydrofolate (n5-methyl-thf) in a reaction requiring methylcobalamin, a coenzyme derived from vitamin b12. the methyl groups transferred from b12 derivative to homocysteine, and cobalamine is recharged from n5-methyl-thf.
b.synthesis of cysteine: homocysteine combines with serine,forming cystathionine, which is hydrolyzed to ?-ketobutyrate and cysteine (see figure). this sequence has the net effect of converting serine to cysteine, and homocysteine to ?-ketobutyrate, which is oxidatively decarboxylated to form propionyl coa. propionyl coa is converted to succinyl coa. because homocysteine is synthesized from the essential amino acid methionine, cysteine is not an essential amino acid as long as sufficient methionine is available.
4. role of homocysteine in vascular disease: elevated plasma homocysteine levels are an independent cardiovascular risk factor that correlates with the severity of coronary artery disease. dietary supplementation with folate, vitamin b12 and vitamin b6-the three vitamins involved in the metabolism of homocystein –leads to a reduction in circulating levels of homocysteine.it is currently unknown if homocysteine-lowering therapy decreases heart disease in the general population. however, the benefits of such therapy can be shown in patients at high risk for vascular disease. for example, vitamin therapy significantly decreases the adverse events, such as re-infarction, in patients undergoing coronary angioplasty, and suggests there is a beneficial effect to reducing homocysteine levels (figure). note also that patients with homocystinuria (characterized by high serum levels of homocysfeline caused by cystathionine synthase deficiency), experience premature vascular disease, and usually die of myocardial infarction, stroke, or pulmonary embolus. thus, there is an association(but not a proven cause and effect relationship) of elevated homocysteine with cardiovascular disease.
f. other amino acids that form succinyl coa
degradation of valine, isoleucine and threonine also results in the production of succinyl coa a- tca cycle intermediate and glucogenic compound.
1. valine and isoleucine are branched-chain amino acids that yield succinyl coa (figure ).
2. threonine is dehydrated to ?-ketobutyrate, which is converted to propionyl coa, the precursor of succinyl coa . [note:threonine can also be converted to pyruvate.]
g. amino acids that form acetyl coa or acetoacetyl coa
leucine, isoleucine, lysine, and tryptophan form acetyl coa or acetoacetyl coa directly, without pyruvate serving as an intermediate(through the pyruvate dehydrogenase reaction)
as mentioned previously, phenylalanine and tyrosine also give rise to acetoacetate during their catabolism (see figure). therefore, there are a total of six ketogenic amino acids.
1. leucine is exclusively ketogenic in its catabolism, forming acetylcoa and acetoacetate (see figure). the initial steps in the catabolism of leucine are similar to those of the other branched-chain amino acids, isoleucine and valine (see below).
2. isoleucine is both ketogenic and glucogenic, because its metabolism yields acetyl coa and propionyl coa. the first three steps in the metabolism of isoleucine are virtually identical to the initial steps in the degradation of the other branched-chain amino acids, valine and leucine (see figure ).
3. lysine, an exclusively ketogenic amino acid, is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. lysine is ultimately converted to acetoacetyl coa.
4. tryptophan is both glucogenic and ketogenic because its metabolism yields alanine and acetoacetyl coa.
catabolism of the branched-chain amino acids
the branched-chain amino acids, isoleucine, leucine, and valine, are essential amino acids.in contrast to other amino acids, they are metabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. because these three amino acids have a similar route of catabolism, it is convenient to describe them as a group.
1. transamination: removal of the amino groups of all three amino acids is catalyzed by a single enzyme, branched-chain ?-amino acid aminotransferase.
2. oxidative decarboxylation: removal of the carboxyl group of the ?-keto acids derived from leucine, valine, and isoleucine is also catalyzed by a single enzyme complex, branched-chain ?-keto acid dehydrogenase complex. this complex uses thiamine pyrophosphate, lipoic acid, fad,nad+ and coenzyme a as its coenzymes. [note: this reaction is similar to the conversion of pyruvate to acetyl coa by pyruvate dehydrogenase, and the oxidation of ?-ketoglutarate to succinyl coa by ?-ketoglutarate dehydrogenase . an inherited deficiency of branched-chain ?-keto acid dehydrogenase results in accumulation of the branched-chain keto acid substrates in the urine. their sweet odor prompted the name maple syrup urine disease.
3. dehydrogenation: oxidation of the products formed in the above reaction yields ?-?-unsaturated acyl coa derivatives.
4. end products: the catabolism of isoleucine ultimately yields acetyl coa and succinyl coa, rendering it both ketogenic and glucogenic. valine yields succinyl coa and is glucogenic. leucine is ketogenic, being metabolized to acetoacetate and acetyl coa.
iv. role of folic acid in amino acid metabolism
some synthetic pathways require the addition of single carbon groups. these "one-carbon units" can exist in a variety of oxidation states. these include methane, methanol, formaldehyde, formic acid, and carbonic acid.it is possible to incorporate carbon units at each of these oxidation states, except methane, into other organic compounds. these single carbon units can be transferred from carrier compounds such as tetrahydrofolic acid and s-adenosylmethionine to specific structures that are being synthesized or modified. the "one-carbon pool" refers to single carbon units attached to this group of carriers.
a. folic acid: a carrier of one-carbon units
the active form of folic acid, tetrahydrofolic acid (thf), is produced from folate by dihydrofolate reductase in a two-step reaction requiring two moles of nadph. the carbon unit carried by thf is bound to nitrogen n5 or n10 or to both n5 and n10.thf allows one-carbon compounds to be recognized and manipulated by biosynthetic enzymes. figure shows the structures of the various members of the thf family, and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate.
v .biosynthesis of nonessential amino acids
nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from essential amino acids. two amino acids- histidine and arginine -are generally classified as nonessential. however, their normal concentrations are limit in, and, during periods of tissue growth (for example, in children or in individuals recovering from wasting diseases), histidine and arginine need to be supplemented in the diet. the synthetic reactions for the nonessential amino acids are described below, and are summarized in figure . [note: some amino acids found in proteins, such as hydroxyproline and hydroxylysine. are modified after their incorporation into the protein.]
a. synthesis from ?-keto acids
alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the ?-keto acids pyruvate, oxaloacetate, and ?-keto-glutarate, respectively. these transamination reactions (figure) are the most direct of the biosynthetic pathways. glutamate is unusual in that it can also be synthesized by the reverse of oxidative deamination catalyzed by glutamate dehydrogenase .
b. synthesis by amidation
1.glutamine: this amino acid, which contains an amide linkage with ammonia at the ?-carboxyl, is formed from glutamate by glutamine synthetase . the reaction is driven by the hydrolysis of atp. in addition to producing glutamine for protein synthesis, the reaction also serves as a major mechanism for the detoxification of ammonia in brain and liver.
2. asparagine: this amino acid, which contains an amide linkage with ammonia at the ?-carboxyl, is formed from aspartate by asparagine synthetase, using glutamine as the amide donor. the reaction requires at, and, like the synthesis of glutamine, has an equilibrium far in the direction of asparagine synthesis.
c. proline
glutamate is converted to proline by cyclization and reduction reactions.
d. serine, glycine, and cysteine
1. serine arises from 3-phosphoglycerate,an intermediate in glycolysis, which is first oxidized to 3-phosphopyruvate, and then transaminated to 3-phosphoserine. serine is formed by hydrolysis of the phosphate ester. serine can also be formed from glycine through transfer of a hydroxymethyl group .
2. glycine is synthesized from serine by removal of a hydroxy-methyl group, also by serine hydroxymethyl transferase .
3. cysteine is synthesized by two consecutive reactions in which homocysteine combines with serine, forming cystathionine, which, in turn, is hydrolyzed to ?-ketobutyrate and cysteine . homocysteine is derived from methionine . because methionine is an essential amino acid, cysteine synthesis can be sustained only if the dietary intake of methionine is adequate.
e. tyrosine
tyrosine is formed from phenylalanine by phenylalanine hydroxylase. the reaction requires molecular oxygen and the coenzyme tetrahydrobiopterin, which can be synthesized by the body. one atom of molecular oxygen becomes the hydroxyl group of tyrosine, and the other atom is reduced to water. during the reaction, tetrahydrobiopterin is oxidized to dihydrobiopterin. tetrahydrobiopterin is regenerated from dihydrobiopterin in a separate reaction requiring nadph. tyrosine, like cysteine, is formed from an essential amino acid and, is therefore, nonessential only in the presence of adequate dietary phenylalanine.
المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
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