The first of them told him so, with the customary prison sign of Death—a raised finger—and they all

added in words, “Long live the Republic!”A Tale of Two Cities. Charles Dickens

Amino acid metabolism

2

Functions of Amino Acid Metabolism• Proteins constantly undergo breakdown and

synthesis• Total protein turnover in a well-fed, adult human

is estimated at about 300 g/day, of which approximately 100 g is myofibrillar protein, 30 g is digestive enzymes, 20 g is small intestinal cell protein, and 15 g is hemoglobin

• The remainder is accounted for by turnover of cellular proteins of various other cells (e.g., hepatocytes, leukocytes, platelets) and a very small amount is lost as free amino acids in urine

• Protein turnover is not completely efficient in the reutilization of amino acids. Some are lost by oxidative catabolism, while others are used in synthesis of non-protein metabolites

• For this reason, a dietary source of protein is needed to maintain adequate synthesis of protein

• There is no distinct storage form for amino acids in the body

• The turnover of some proteins, particularly those in muscle, is increased under conditions of fasting and starvation

3

• Plants and some bacteria synthesize all 20 amino acids. Humans (and other animals) can synthesize some (the non-essential amino acids) but require the others to be supplied by the diet (the essential amino acids)

• The eight essential amino acids are isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, and valine

• Under certain conditions, some nonessential amino acids may become essential –these amino acids are known as conditionally essential amino acids

• Although arginine and histidine are not essential amino acids in adults, their rates of synthesis in neonates are not adequate to meet their requirements for optimal growth; they should, therefore, be supplied in the diet

• Synthesis of cysteine and tyrosine is dependent on adequate dietary intake of methionine and phenylalanine, respectively

4

• Glutamine, a nitrogen donor in the synthesis of purines and pyrimidines required for nucleic acid synthesis, aids in growth, repair of tissues, and promotion of immune function

• Enrichment of glutamine in nutrition augments recovery of seriously ill patients

• Exogenous arginine also becomes essential in cases of sepsis (the presence of various pathogenic organisms, or their toxins, in the blood or tissues), when there is both a decrease in endogenous synthesis of arginine and an increased requirement of arginine for the synthesis of protein and nitric oxide

• For protein synthesis to occur, all amino acids must be present in sufficient quantities. Absence of any one essential amino acid leads to cessation of protein synthesis, catabolism of unused amino acids, increased loss of nitrogen in urine, reduced growth

• Negative nitrogen balance exists when the amount of nitrogen lost from the body (as nitrogen metabolites excreted in urine and feces) exceeds that taken in

5

•Negative nitrogen balance occurs in malabsorption syndromes, fever, trauma, cancer, and excessive production of catabolic hormones•When the dietary nitrogen intake equals nitrogen losses, the body is in nitrogen balance. In normal adults, anabolism equals catabolism•When nitrogen intake exceeds nitrogen losses, there is a positive nitrogen balance, with anabolism exceeding catabolism. In this case, the body retains nitrogen as tissue protein, which is a characteristic of active growth and tissue repair• So, in general, there are three major fates for

amino acids:1.Synthesis of new proteins for growth or repair2.Synthesis of a range of nitrogen-containing small

compounds3.Catabolism. This results, eventually, in formation of

ammonia and small carbon-containing compounds. The carbon skeletons are used for the synthesis of glucose and triacylglycerol or for energy production.

The ammonia is converted to urea

6

Protein Digestion • The digestion of proteins begins in the stomach and

is completed in the intestine • The enzymes that digest proteins are produced as

inactive precursors (zymogens) • The inactive zymogens are secreted from the cells

in which they are synthesized and enter the lumen of the digestive tract, where they are cleaved to smaller forms that have proteolytic activity

• These active enzymes have different specificities; no single enzyme can completely digest a protein

• However, by acting in concert, they can digest dietary proteins to amino acids and small peptides, which are cleaved by peptidases associated with intestinal epithelial cells

• The acid in the stomach lumen alters the conformation of pepsinogen so that it can cleave itself, producing the active protease pepsin

7

• Thus, the activation of pepsinogen is autocatalytic• Dietary proteins are denatured by the acid in the

stomach. This serves to inactivate the proteins and partially unfolds them such that they are better substrates for proteases

• However, at the low pH of the stomach, pepsin is not denatured and acts as an endopeptidase, cleaving peptide bonds at various points within the protein chain

• Although pepsin has a fairly broad specificity, it tends to cleave peptide bonds in which the carboxyl group is provided by an aromatic or acidic amino acid

• A proteolytic enzyme secreted by gastric mucosa of infants is chymosin (rennin), which functions to clot milk and promote its digestion by preventing rapid passage from the stomach

• Chymosin hydrolyzes casein, a mixture of several related milk proteins, to paracasein, which reacts with Ca 2+ to yield the insoluble curd

• As the gastric contents empty into the intestine, they encounter the secretions from the exocrine pancreas

8

• One of these secretions is bicarbonate, which, in addition to neutralizing the stomach acid, raises the pH such that the pancreatic proteases, which are also present in pancreatic secretions, can be active

• These pancreatic proteases are also secreted as zymogens. Because the active forms of these enzymes can digest each other, it is important for their zymogen forms all to be activated within a short span of time

• Trypsin, elastase, and chymotrypsin are endopeptidases. Carboxypeptidases are exopeptidases

• The combined action of these enzymes produces oligopeptides having two to six amino acid residues and free amino acids

• Exopeptidases produced by intestinal epithelial cells act within the brush border and also within the cell

• Aminopeptidases, located on the brush border, cleave one amino acid at a time from the amino end of peptides. Intra-cellular peptidases act on small peptides that are absorbed

9

Th

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Absorption of Amino Acids• Amino acids are absorbed from the lumen of the

small intestine principally by semi-specific Na+-dependent transport proteins in the luminal membrane of the intestinal cell brush border, similar to that already seen for carbohydrate transport

• At least six different Na+-dependent amino acid carriers are located in the apical brush border membrane of the epithelial cells

• These carriers have an overlapping specificity for different amino acids (for neutral amino acids, proline and hydroxyproline, …)

• As with glucose transport, the Na+-dependent carriers of the apical membrane of the intestinal epithelial cells are also present in the renal epithelium

• The amino acids are then transported out of the cell into the portal circulation principally by facilitated transporters in the serosal membrane

11

• Di- and tripeptides enter the epithelial cells through symport with H+. The H+ gradient is maintained by the Na+ - H+ exchanger

• Amino acids that enter the blood are transported across cell membranes of the various tissues principally by Na+-dependent cotransporters and, to a lesser extent, by facilitated transporters

• In this respect, amino acid transport differs from glucose transport, which is Na+-dependent transport in the intestinal and renal epithelium but facilitated transport in other cell types

12

Disorders of Protein Malnutrition, Digestion and Amino Acid Absorption

• The principal causes of protein maldigestion and malabsorption are diseases of the exocrine pancreas (such as cystic fibrosis) and small intestine

• Defects in neutral amino acid transport (Hartnup disease), in basic amino acids and cystine (cystinuria),… have been reported

• The clinical severity of these disorders is usually minimal and relates to the loss of amino acids or relative insolubility of certain amino acids in the urine

• Kwashiorkor (in Ga, "the disease the first child gets when the second is on the way") is a form of protein-calorie malnutrition that is caused by dietary protein deficiency and is often exacerbated by infection

• The classic presentation, particularly in poorer countries, is a young child who has been weaned to an adult diet that lacks sufficient protein to sustain healthy growth

13

•The characteristics of kwashiorkor include growth failure, edema, fatty liver, and “flaky paint” patches of skin• Because of the low protein intake, there is a deficiency of amino acids for synthesis of serum albumin and other plasma proteins, resulting in edema and the characteristic swollen abdomen and limbs•The situation is made worse by the availability of ample dietary carbohydrates, which stimulate insulin secretion and thus inhibit mobilization of amino acids from skeletal muscle•This dietary carbohydrate also provides substrate for fatty acid synthesis, which in the absence of adequate protein synthesis results in fatty liver and hepatomegaly• The clinical manifestation of a diet deficient in

both protein and energy is marasmus, (from the Greek "to waste away") which results in severe muscle wasting and marked growth retardation

14

Protein Turnover• The amino acid pool within cells is generated both

from dietary amino acids and from the degradation of existing proteins within the cell

• All proteins within cells have a half-life (t1/2), a time at which 50% of the protein that was synthesized at a particular time will have been degraded

• The rates of protein turnover vary enormously, depending on the nature of the protein, the condition of the subject and the tissue

• Proteins (mainly enzymes) in the liver are replaced every few hours or days whereas structural proteins (e.g. collagen, contractile proteins) are stable for several months

• Why should turnover occur?1. It helps remove defective proteins and replace

them with normal ones 2.The regulation of hormone and enzyme levels

15

• There are two main pathways for protein degradation: the lysosomal-autophagic system and the ubiquitin-proteasome degradation pathway

• The quantitative importance of each pathway varies from one tissue to another and from one protein to another

• Although hydrolysis of the peptide bonds does not involve ATP, the various processes of protein degradation require considerable expenditure of energy, possibly more than is required for protein synthesis

• Protein turnover contributes at least 20% to resting energy expenditure (basal metabolic rate)

The Lysosomal –Autophagic System• In general, extracellular, membrane-

associated, and long-lived intracellular proteins are degraded in lysosomes by ATP-independent processes

16

• A variety of proteases known as cathepsins and peptidases exist so that proteins can be hydrolysed completely to amino acids

• The pH within the lysosome is 4.5–5.0 and all lysosomal enzymes exhibit low pH optima

• This ensures that, if they leak into the cytosol, their activity is very low and little damage is done

• This low pH within the organelle is maintained by a proton pump, driven by the hydrolysis of ATP, thus contributing to the energy

• Proteins enter the lysosome by two main mechanisms: Vesicles transport extracellular particles and

membrane proteins into the cell, where they fuse with the lysosomes (endocytosis)

The endoplasmic reticulum engulfs some cytosolic proteins to form vesicles which fuse with the lysosomes

17

Lysosomal Degradation

18

The Ubiquitin-Proteasome System• In general, the degradation of regulatory proteins

with short half-lives and of abnormal proteins occurs in the cytosol, through the ubiquitin-proteasome system

• This system is quantitatively the most important process for protein breakdown in mammalian cells. It is so named because it involves the proteolytic enzyme (the proteasome), and the protein ubiquitin

• The proteasome is a very large complex of at least 50 subunits. It is present in a wide variety of tissues and can constitute up to 1% of soluble protein in a cell

• The catalysis occurs within the central core of the molecule and ATP hydrolysis is required to ‘drive’ the protein into the core.

• Before the complex can break down proteins, the protein must first be ‘tagged’ by complexing with ubiquitin

• Ubiquitin, so named because it is present in all eukaryotic cells, is a small (8.5 kDa, 76 aa residues) protein

19

• The protein substrate has amino groups in the side chains of its Lys residues

• Ubiquitin has got a C-terminal Gly. The carboxyl group of this Gly forms an isopeptide bond with the amino group of Lys

• Oftentimes, the target protein is polyubiquitinylated, in which additional ubiquitin molecules are added to previous ubiquitin molecules

• The Lys on one ubiquitin molecule serve as internal acceptors for the carboxyl of Gly on another ubiquitin molecule, allowing the formation of a chain

• The residue present at its amino terminal affects whether a protein is ubiquitinated. Amino terminal Met or Ser retards, whereas Asp or Arg accelerates ubiquitination

• In addition, many proteins that contain regions rich in the amino acids proline (P), glutamate (E), serine (S), and threonine (T) have short half-lives. These regions are known as PEST sequences

20

• Three enzymes are involved in the attachment of ubiquitin to a protein: E1 (an activating enzyme), E2 (a conjugating enzyme), and E3 (a ligase)

• Some pathological conditions vividly illustrate the importance of the regulation of protein turnover. For example, human papilloma virus (HPV) encodes a protein that activates a specific E3 enzyme. The enzyme ubiquitinates the tumor suppressor p53 and other proteins that control DNA repair, which are then destroyed. The activation of this E3 enzyme is observed in more than 90% of cervical carcinomas

21

• The proteasome degrades the targeted protein, releasing intact ubiquitin that can again mark other proteins for degradation

• The basic proteasome is a cylindrical 20S protein complex with multiple internal proteolytic sites

• ATP hydrolysis is used both to unfold the tagged protein and to push the protein into the core of the cylinder

• Additional subunits, some of which catalyze ATP hydrolysis, form a cap which adds to one or both ends of a 20S proteasome to give a larger 26S proteasome.

• Different cap complexes alter the specificity of the proteasome

• For example, the PA700 cap is required for ubiquitinated proteins, whereas the PA28 cap targets only short peptides to the complex

• After the target protein is degraded, the resultant amino acids join the intracellular pool of free amino acids

22

The Proteasome

23

• Some key intracellular processes, which involve proteasomal degradation include:

The cell cycle • The concentrations of specific proteins known as

cyclins (that regulate the cell cycle) is regulated by synthesis and degradation

Transcription factors • These factors activate the expression of genes. In

order to carry out their regulatory function, they must have short half lives. Their degradation is carried out by this system

Formation of antigens from the degradation of pathogens

• The proteolytic system hydrolyses proteins of pathogens that are present within the host cell to produce a short peptide which forms a complex with a specific protein, known as the major histocompatibility complex (MHC) protein

• The peptide is, in fact, the antigen

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• At the plasma membrane, the MHC protein locates within the membrane and the small peptide sits on the outside of the membrane, where it can interact with the receptor on T- lymphocytes

Defects in Protein Degradation and Diseases • The importance of the proteasomal-ubiquitin

system in the degradation of cellular proteins or proteins of pathogens suggests that any defects in this system could result in disease

• Prion diseases and amyloid diseases (such as Alzheimer’s and Parkinson’s) involve the aggregation of degradation-resistant proteins

• Failure to control the rate of degradation of cyclins could lead to their over-expression, increasing the risk of tumour development

• Infectious agents may hijack cell machinery involved in ubiquitination and protein degradation system

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Interorgan Relation in Amino Acid Metabolism• In the fed state, amino acids released by digestion

of dietary proteins (except the greater portion of branched-chain amino acids) travel through the hepatic portal vein to the liver, where they are used for the synthesis of proteins, particularly the blood proteins, such as serum albumin

• Excess amino acids are converted to glucose or to triacylglycerols. The latter are then packaged and secreted in VLDL. The glucose produced from amino acids in the fed state is stored as glycogen or released into the blood if blood glucose levels are low

• Amino acids that pass through the liver are converted to proteins in cells of other tissues

• Muscle generates over half of the total body pool of free amino acids in the post-absorptive state, and liver is the site of the urea cycle enzymes necessary for disposal of excess nitrogen

26

• Muscle and liver thus play major roles in maintaining circulating amino acid levels

• Free amino acids, particularly alanine and glutamine, are released from muscle into the circulation. Alanine, which appears to be the vehicle of nitrogen transport in the plasma, is extracted primarily by the liver

• Glutamine is extracted by the gut and the kidney (among other tissues), both of which convert a significant portion to alanine

• The kidney provides a major source of serine for uptake by peripheral tissues, including liver and muscle

• Branched-chain amino acids, particularly valine, are released by muscle and taken up predominantly by the brain

Metabolism of Ammonia• Transamination is the major process for removing

nitrogen from amino acids. In most instances, the nitrogen is transferred as an amino group from the original amino acid to α- ketoglutarate, forming glutamate

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Fed State Post-absorptive State

28

• The original amino acid is converted to its corresponding α -keto acid

• For example, the amino acid aspartate can be transaminated to form its corresponding α-keto acid, oxaloacetate

• In the process, the amino group is transferred to α-keto-glutarate, which is converted to its corresponding amino acid, glutamate

• All amino acids except lysine, threonine and proline undergo transamination reactions

• The enzymes catalyzing these reactions are known as transaminases or aminotransferases

• Pyridoxal phosphate (PLP), is the cofactor in transamination reactions

• The aldehyde group of PLP can accept the α-amino group from an amino acid, generating pyridoxamine phosphate, which in turn donates that amino group to an α-ketoacid, regenerating PLP

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The Mechanism of Transamination Reactions

30

• The activated intermediate in this process is a Schiff base

• PLP-containing enzymes also catalyze many other reactions involving amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters; PLP is also involved in the glycogen phosphorylase reaction

• Because transamination reactions are readily reversible, they can be used to remove nitrogen from amino acids or to transfer nitrogen to α-keto acids to form amino acids

• Thus, they are involved both in amino acid degradation and in amino acid synthesis

• Liver is the major site of aminotransferase activity• The two principal liver transaminases are alanine

aminotransferase (ALT), which catalyzes the reaction alanine + α-ketoglutarate → pyruvate + glutamate and aspartate aminotransferase (AST), which catalyzes the reaction aspartate + α-ketoglutarate → oxaloacetate + glutamate

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• Most of the NH4+ generated when muscle proteins

are broken down during a fast is exported in the form of alanine

• In the liver, ALT catalyzes the transamination of alanine, generating pyruvate which can be utilized for gluconeogenesis, and glutamate which provides nitrogen atoms for urea synthesis

• Some of the glutamate nitrogen is released as ammonium ions by the enzyme glutamate dehydrogenase

• Concurrently, AST utilizes some of the glutamate to generate aspartate by transfer of the amino group from glutamate to oxaloacetate

• The NH4+ from the glutamate dehydrogenase

reaction and the aspartate from the AST reaction provide the two nitrogens for urea synthesis

• Aminotransferases are intracellular enzymes that have both cytosolic and mitochondrial isoforms

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• When there is liver damage, as occurs with cirrhosis or viral hepatitis, these aminotransferase are released from the hepatocytes

• Increased plasma levels of ALT and AST are thus markers of liver damage

• In the older clinical literature, these enzymes are sometimes referred to as SGPT (serum glutamate:pyruvate transaminase) and SGOT (serum glutamate:oxaloacetate transaminase), respectively

Generation of Ammonium Ions from Amino Acids

• Two steps are required to generate ammonium ions from most of the common amino acids

• The first is the aminotransferase reaction and the second step is the NAD+/NADP+-dependent oxidative deamination of glutamate, which releases a free ammonium ion, regenerating α-ketoglutarate in the process

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• This reaction, which occurs in the mitochondria of most cells, is readily reversible; it can incorporate ammonia into glutamate or release ammonia from glutamate

• The nitrogen in glutamate can be given off for biosynthesis or the removal in the form of urea

• In general, glutamate can be thought of as a reservoir for amino groups while alanine and glutamine are the major transport forms of nitrogen in the blood

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• In addition to glutamate, a number of amino acids release their nitrogen as NH4

+

• Histidine may be directly deaminated to form NH4+

and urocanate• The deaminations of serine and threonine are

dehydration reactions that require pyridoxal phosphate and are catalyzed by serine and threonine dehydratases, respectively

• Serine forms pyruvate, and threonine forms α-ketobutyrate. In both cases, NH4

+ is released• Glutamine and asparagine contain R group amides

that may be released as NH4+ by deamidation

• Asparagine is deamidated by asparaginase, yielding aspartate and NH4

+ . Glutaminase acts on glutamine, forming glutamate and NH4

+ • The glutaminase reaction is particularly important

in the kidney, where the ammonium ion produced is excreted directly into the urine as a buffer

35

• Ammonium ion is also generated through the deamination of AMP to IMP (inosine monophosphate) by adenosine monophosphate deaminase

• This reaction is especially active in exercising muscle which generates AMP. When ATP levels are low, muscle can generate additional ATP directly from ADP by means of the (adenylate kinase reaction

• Removal of the resulting AMP is necessary if the reaction is to continue

• The pathway by which AMP is deaminated to IMP and IMP is subsequently utilized for resynthesis of AMP is referred to as the purine nucleotide cycle

• This cycle is also active in the brain but not in the liver

• D-amino acids from bacterial cell walls and cooked food are metabolized by D-amino acid oxidase that is active in the liver and the kidneys; the products are a keto acid, FADH2 and NH4

+

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• Bacteria in the gut also act on amino acids, urea and other nitrogen containing molecules to release free ammonia

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The Urea Cycle • Humans excrete excess dietary nitrogen primarily

as urea in the urine• At plasma concentrations greater than 50 μM,

ammonia (at physiological pH, 98.5% exists as NH4+

) is toxic to the CNS • Different animals excrete excess nitrogen as

ammonia, uric acid, or urea• The aqueous environment of fish, which are

ammonotelic (excrete ammonia), compels them to excrete water continuously to facilitate excretion of the highly toxic molecule ammonia

• Birds, which must conserve water and maintain low weight, are uricotelic and excrete uric acid as semisolid guano

• Many land animals, including humans, are ureotelic and excrete nontoxic, water-soluble urea

• The urine of humans contains nitrogenous compounds other than urea, including uric acid, creatinine, and ammonia

38

• These molecules serve other, distinct functions or represent breakdown products of certain metabolites

• For example, creatinine is a breakdown product of muscle creatine phosphate and, as such, provides the clinician with a convenient measure of muscle mass

• Uric acid is the end product of purine catabolism• The kidney also excretes some nitrogen directly in

the form of ammonium ions, which serve to buffer acidic, anionic waste products such as β-hydroxybutyrate, acetoacetate, and sulfate

• Excretion of ammonium ions is thus increased during ketoacidosis and other metabolic conditions where excess organic acids are produced

The Reactions of the Urea CycleSynthesis of Carbamoyl Phosphate • In the first step of the urea cycle, NH4

+ , bicarbonate, and ATP react to form carbamoyl phosphate. The cleavage of 2 ATPs is required to form the high-energy phosphate bond of CP

39

• The enzyme that catalyzes this reaction is carbamoyl phosphate synthetase I (CPS I), and it is localized to mitochondria

• Carbamoyl phosphate synthetase II (CPS II) is a cytosolic enzyme that generates carbamoyl phosphate for pyrimidine synthesis. The nitrogen donor in this case is glutamine

The Synthesis of Citrulline • Orinthine transcarbamoylase catalyzes the

transfer of the carbamoyl group from carbamoyl phosphate to the amino group in the side chain of the amino acid ornithine, generating citrulline. The high- energy phosphate bond of carbamoyl phosphate provides the energy required for this reaction, which occurs in mitochondria

40

• Citrulline is transported out of the mitochondrion in exchange for ornithine (antiport)

Formation of Argininosuccinate • In the cytosol, citrulline reacts with aspartate, the

second source of nitrogen for urea synthesis, to produce argininosuccinate

• This reaction, catalyzed by argininosuccinate synthetase, is driven by the hydrolysis of ATP to AMP and PPi

• The reaction is driven forward by hydrolysis of pyrophosphate to inorganic phosphate

41

Formation of Arginine and Fumarate • Argininosuccinate is cleaved by argininosuccinate

lyase to form fumarate and arginine • Fumarate is produced from the carbons of

argininosuccinate provided by aspartate• Fumarate is converted to malate (using

cytoplasmic fumarase), which is used either for the synthesis of glucose by the gluconeogenic pathway or for the regeneration of oxaloacetate by cytosolic malate dehydrogenase

42

• The oxaloacetate that is formed is transaminated to generate the aspartate that carries nitrogen into the urea cycle

• Thus, the carbons of fumarate can be recycled to aspartate

The Krebs Bicycle

43

• When these microorganisms die, their proteins are digested, releasing the amino acids to be absorbed into the blood

• Some of the ammonia produced by urease travels to the liver and is converted back to urea

• Ornithine generated by arginase is transported back into mitochondria to continue the cyclic process of urea synthesis

• The urea cycle in effect changes ornithine to arginine

Urea Salvage

44The Urea Cycle

45

• Some of the enzymes of the urea cycle are present in tissues other than the liver

• The intestinal mucosa can convert ornithine to citrulline, and the kidney can convert the resultant citrulline to arginine

• However, since the kidney and intestine lack arginase, they cannot synthesize urea

Regulation of Urea Synthesis• In general, the urea cycle is regulated by substrate

availability: the higher the rate of ammonia production, the higher the rate of urea formation

• Regulation by substrate availability is a general characteristic of disposal pathways, such as the urea cycle, which remove toxic compounds from the body

• Two other types of regulation control the urea cycle: allosteric activation of CPSI by N-acetylglutamate (NAG) and induction/repression of the synthesis of urea cycle enzymes

46

• NAG is formed specifically to activate CPSI; it has no other known function in mammals (just like F 2,6-BP)

• The synthesis of NAG from acetyl-CoA and glutamate is stimulated by arginine

• Thus, as arginine levels increase within the liver, two important reactions are stimulated

• The first is the synthesis of NAG, which will increase the rate at which carbamoyl phosphate is produced

• The second is to produce more ornithine (via the arginase reaction), such that the cycle can operate more rapidly

• The induction of urea cycle enzymes occurs in response to conditions that require increased protein metabolism, such as a high-protein diet or prolonged fasting

• In both of these physiologic states, as amino acid carbon is converted to glucose, amino acid nitrogen is converted to urea

• Stress (like in the case of sepsis, burns or trauma) also induces urea cycle enzymes

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Hyperammonemia and Treatment•The finding of elevated blood levels of ammonia is evidence that the conversion of ammonia to urea is impaired in some way•Hyperammonemia in adults is usually the consequence of impaired liver function, secondary to liver disease (e.g., cirrhosis), organ transplantation, or chemotherapy• Transient hyperammonemia is often seen in premature neonates with immature liver function and/or inadequate hepatic blood flow

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• Impaired urea synthesis may also be the result of a genetic defect in one of the enzymes of the urea cycle• Regardless of its etiology, hyperammonemia is usually accompanied by increased plasma levels of glutamine, the amino acid that the brain uses as a vehicle to export excess ammonium ions•Ammonia is toxic to the central nervous system, where it can cause both acute encephalopathy and long-term irreversible brain damage; however, the pathophysiologic mechanisms are not fully understood• One possible cause is the increased synthesis of the neurotransmitters glutamate and GABA and subsequent derangements of neurotransmission•Another possible mechanism for ammonia toxicity in the brain involves the depletion of TCA-cycle intermediates by diversion of α-ketoglutarate to glutamate and glutamine synthesis, which would compromise the ability of the neural cells to generate ATP

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• Treatment for hyperammonemia involves dialysis to remove the excess ammonia

• In acute cases, oral sodium benzoate and sodium phenylbutyrate are sometimes administered to provide alternate pathways for nitrogen excretion as hippurate and phenylacetylglutamine, respectively

• The body then has to use its nitrogen to resynthesize the excreted amino acid

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• Protein intake should also be severely restricted in patients with hyperammonemia

• At the same time, it is important to provide adequate intake of carbohydrates to minimize further catabolism of endogenous protein

• Neonatal hyperammonemia is due to inborn errors of urea-cycle enzymes

• In these defects, low blood urea nitrogen (BUN) accompanies the hyperammonemia

• The most common inborn error of the urea cycle is a deficiency of ornithine transcarbamoylase, an X-linked disorder

• Ammonia intoxication is most severe when the metabolic block occurs at CPS I or OTC, because if citrulline can be synthesized, some ammonia has already been removed by being covalently linked to an organic metabolite

• If the enzyme defect is in argininosuccinate lyase, massive arginine supplementation is beneficial

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• Once argininosuccinate has been synthesized, the two nitrogen molecules destined for excretion have been incorporated into the substrate; the problem is that ornithine cannot be regenerated

• If ornithine could be replenished to allow the cycle to continue, argininosuccinate could be used as the carrier for nitrogen excretion from the body

• The deficiencies of CPS I, OTC, arginase (and argininosuccinate synthase ) are treated with benzoate and phenylbutyrate

• NAG synthase deficiency cannot be treated by administration of NAG, since NAG undergoes cytosolic inactivation by deacylation and is not readily permeable across the inner mitochondrial membrane

• An analogue of NAG, N-carbamoylglutamate, activates CPSI, does not share the undesirable properties of NAG, and has been effective in the management of this deficiency

Elevated BUN is an indication of a post-hepatic failure in nitrogen excretion

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The Origin and Fate of Carbon Skeletons of Amino Acids

• As amino acids are degraded, their carbons are converted to:

(a) CO2 (b) compounds that produce glucose in the liver (pyruvate and the TCA cycle intermediates α-ketoglutarate, succinyl CoA, fumarate, and oxaloacetate), and

(c) ketone bodies or their precursors (acetoacetate and acetyl CoA)

• Degradative pathways may also directly provide NADH & FADH2

• For simplicity, amino acids are considered to be glucogenic if their carbon skeletons can be converted to a precursor of glucose and ketogenic if their carbon skeletons can be directly converted to acetyl CoA or acetoacetate

• Some amino acids contain carbons that produce a glucose precursor and other carbons that produce acetyl CoA or acetoacetate

• These five amino acids are both glucogenic and ketogenic; lysine and leucine are strictly ketogenic

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Glucogenic

Amino Acids

54

Ketogenic

Amino Acids

• The carbon skeletons of 10 non-essential amino acids may be produced from glucose through intermediates of glycolysis or the TCA cycle

• The nitrogen comes from another amino acid or ammonia

• The 11th non-essential amino acid, tyrosine, is synthesized by hydroxylation of the essential amino acid phenylalanine

• Only the sulfur of cysteine comes from the essential amino acid methionine; its carbons and nitrogen come from serine

Intermediates of amino acid degradation and synthesis may overlap (e.g. pyruvate, oxaloacetate, α-ketoglutarate)

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The Precursors for Non-Essential Amino

Acids

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Amino Acid Metabolism and AbnormalitiesGlycine •The conversion of glycine to glyoxylate by the enzyme D-amino acid oxidase is a degradative pathway of glycine that is medically important• Once glyoxylate is formed, it can be oxidized to oxalate, which is sparingly soluble and tends to precipitate in kidney tubules, leading to kidney stone formation•Approximately 40% of oxalate formation in the liver comes from glycine metabolism•Dietary oxalate accumulation has been estimated to be a low contributor to excreted oxalate in the urine because of poor absorption of oxalate in the intestine •Crystals of calcium oxalate account for up to 75% of all kidney stones

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Branched-Chain Amino Acids • Although much of the catabolism of amino acids

takes place in the liver, the three amino acids with branched side chains (leucine, isoleucine, and valine) are oxidized as fuels primarily in muscle, adipose, kidney, and brain tissue

• These extrahepatic tissues contain an aminotransferase, absent in liver, that acts on all three branched-chain amino acids to produce the corresponding α-keto acid

• Oxidative decarboxylation of the α-keto acids is catalyzed by a branched-chain keto acid dehydrogenase (BCKADH) complex analogous to that of PDC and α-ketoglutarate dehydrogenase complexes. BCKADH is widely distributed in mammalian tissue mitochondria

• There is a relatively rare genetic disease in which the three branched-chain - α keto acids (as well as their precursor amino acids, especially leucine) accumulate in the blood and “spill over” into the urine

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• This condition, called maple syrup urine disease because of the characteristic odor (like burnt sugar) imparted to the urine by the α-keto acids, results from a defective BCKADH complex

• Untreated, the disease results in abnormal development of the brain, mental retardation, and death in early infancy

• Treatment entails rigid control of the diet, limiting the intake of valine, isoleucine, and leucine to the minimum required to permit normal growth

• The deficiency of thiamine could also lead to the accumulation of branched-chain - α keto acids

Phenylalanine • Phenylalanine is the immediate precursor of

tyrosine. The reaction is catalyzed by phenylalanine hydroxylase with tetrahydrobiopterin (BH4) as cofactor

• This pathway provides tyrosine for both protein synthesis and the synthesis of catecholamines and thyroid hormones. It is also the major pathway for the catabolism of excess phenylala.

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Branched-Chain Amino

Acid Metabolism

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• BH4 is a cofactor for the hydroxylation of aromatic amino acids

• Unlike so many of the other cofactors in intermediary metabolism, BH4 is not a vitamin

• Instead, it is synthesized from GTP

• Phenylalanine hydroxylase is a mixed-function oxidase, which simultaneously oxidizes phenylalanine and removes two hydrogen atoms from BH4

• The resulting BH2 is then recycled to BH4, with NADH serving as the reductant

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• Phenylketonuria (PKU) is a relatively common inborn error of amino acid metabolism

• People with PKU lack phenylalanine hydroxylase• Lacking phenylalanine hydroxylase activity,

plasma phenylalanine increases to the point where phenylalanine is metabolized by phenylalanine transaminase, with a resulting plasma accumulation and urinary excretion of phenylpyruvate, phenylacetate, and other metabolites of phenylalanine

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• Untreated, severe forms of PKU result in progressive and severe mental retardation, with other neurological manifestations

• PKU is one of a number of genetic diseases in which neonatal screening and rapid medical intervention, ideally within the first week of life, result in successful outcomes

• Treatment involves severe restriction of dietary phenylalanine, which requires elimination of protein-rich foods

• Some people with PKU have a deficit in the ability to either synthesize or recycle BH4, and therefore also have impaired synthesis of catecholamines and serotonin

• Administration of BH4 is effective in treating the hyperphenylalanemia of these patients; however, since BH4 does not cross the blood-brain barrier, this therapy does not restore neurotransmitter synthesis in the CNS

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• Once phenylalanine has been changed into tyrosine, tyrosine could follow a degradative pathway to produce fumarate and acetoacetate

• The deficiencies of enzymes involved in this pathway lead to certain abnormalities: Deficiency of tyrosine aminotransferase –

tyrosinemia II Deficiency of fumarylacetoacetate hydrolase –

tyrosinemia I Deficiency of homogentisate oxidase –

alcaptonuria • In alcaptonuria, homogentisate is eliminated in

urine, which darkens upon exposure to air owing to oxidation of homogentisate

• Later in life, the chronic accumulation of homogentisate in cartilage may cause arthritic joint pain

• Alcaptonuria is of considerable historical interest. Archibald Garrod discovered in the early 1900s that this condition is inherited, and he traced the cause to the absence of a single enzyme; the first identified inborn error of metabolism

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• Many reactions in human metabolism involve the transfer of an activated one-carbon group from a donor molecule to an acceptor molecule

• Some of these reactions function in catabolic pathways, for example in the breakdown of serine and histidine, whereas others occur in anabolic processes such as in the pathway of purine synthesis or the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP)

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• One-carbon units can exist in various oxidation states. The most oxidized form, CO2, is transferred by biotin.

• One-carbon groups at lower levels of oxidation than CO2 are transferred by reactions involving tetrahydrofolate (FH4), vitamin B12, and S-adenosylmethionine (SAM)

• Although reactions involving one-carbon transfer occur in essentially all cells, they are especially prominent in the liver which is the major site of purine synthesis

• Relatively high levels of enzymes that use FH4are also found in the brain, where one-carbon groups are used to maintain the pool of SAM for the methylation reactions involved in both catecholamine synthesis and inactivation as well as to synthesize BH4, the cofactor for hydroxylation reactions of catecholamine and serotonin synthesis

• One-carbon metabolism plays an important in the synthesis of purines that are components of RNA and DNA and in the generation of thymidylate for DNA synthesis

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• It is therefore most active during periods of rapid cellular growth, including embryogenesis and early postnatal development, and in rapidly dividing cells such as the intestinal epithelium and stem cells of both the erythropoietic and immune cell lineages

Tetrahydrofolate • The vitamin folate was named for its presence in

green, leafy vegetables (foliage)• Although humans can synthesize all of the

components of the vitamin (glutamate, pteridine and para-aminobenzoic acid (PABA)), they lack the enzyme required to join PABA to the pteridine ring

• Enzymes on the brush border of the intestine cleave off glutamate residues from dietary folates to give the monoglutamate form of folate, which is then absorbed

• Inside the enterocytes, this folate is changed to FH4 by two successive reduction steps

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• The reduction steps are catalyzed by dihydrofolate reductase, and use NADPH as the reductant• After absorption from the intestine and transport of

FH4 to the liver and other cells, the polyglutamate chain is restored by polyglutamate synthetase, trapping the active form of the cofactor within the cell• Subsequent release of the vitamin from hepatic

stores into the blood requires hydrolysis of these additional glutamate residues• One-carbon groups transferred by FH4 are attached

either to nitrogen N5 or N

10 or they form a bridge

between N5 and N

10

• Groups that can be attached to N5 include formyl (-

CHO), formimino (-CH=NH), or methyl (-CH3 ) groups• N

10 can carry formyl groups

• Methylene (-CH2- ) or methenyl (-CH=) groups

form bridges between N5 and N

10

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The

Synthesis of

FH4

• The collection of one-carbon groups attached to FH4 is known as the one-carbon pool• While attached to FH4 ,

these one-carbon units can be oxidized and reduced. Thus, reactions requiring a carbon at a particular oxidation state may use carbon from the one-carbon pool that was donated at a different oxidation state• Once the methyl group is

formed, it is not readily reoxidized back to N

5, N

10

methylene FH4, and thus

N5-methyl-FH4 will tend

to accumulate in the cell

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• Carbon sources for the one-carbon pool include serine, glycine, formaldehyde, histidine, and formate • Serine is the major carbon

source of one-carbon groups in the human• Its hydroxymethyl group is

transferred to FH4 in a reversible reaction• This reaction produces

glycine and N5, N

10 -

methylene- FH4 • Because serine can be

synthesized from 3-phosphoglycerate, dietary carbohydrate can serve as a source of carbon for the one-carbon pool

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• Delivery of one-carbon groups is involved in the synthesis of glycine from serine (because the conversion of serine to glycine is readily reversible), the synthesis of the base thymine required for DNA synthesis, the purine bases required for both DNA and RNA synthesis, and the transfer of methyl groups to vitamin B12• After the carbon group carried by FH4 is reduced to

the methyl level, it is transferred to vitamin B12. This is the only reaction through which the methyl group can leave FH4

• Lack of vitamin B 12 to regenerate free FH4 will

“trap” the FH4 as N5 -methyl- FH4, thereby limiting

the availability of FH4 for other biosynthetic reactions

Vitamin B12• Utilization of dietary vitamin B12 is dependent on

both gastric HCl and two specialized proteins, R proteins and intrinsic factor. • Dietary vitamin B 12 is covalently bound to

polypeptides; release of vitamin B 12 normally occurs in the stomach through the combined hydrolytic actions of HCl and pepsin

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• R proteins (also designated haptocorrins or cobalophilins) are present in both saliva and gastric juice• They bind the vitamin B12 prior to its release from

the polypeptides, and remain associated with vitamin B12 until the R proteins are hydrolyzed in the small intestine• Intrinsic factor (IF), a glycoprotein produced by the

parietal cells of the stomach, is essential for the absorption of vitamin B12• Intrinsic factor is so named because early studies

demonstrated that both a dietary (extrinsic) factor and a protein produced by the normal stomach (intrinsic) were necessary for the prevention of pernicious anemia• As soon as vitamin B12 is released from the R

proteins, it binds to intrinsic factor• The intrinsic factor–B12 complex attaches to

specific receptors the ileum, after which the complex is internalized• The B12 within the enterocyte complexes with

transcobalamin II and then is released into circulation

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• The transcobalamin II–B12 complex delivers B12 to the tissues, which contain specific receptors for this complex• The liver takes up

approximately 50% of the vitamin B12, and the remainder is transported to other tissues• The amount of the

vitamin stored in the liver is large enough that 3 to 6 years pass before symptoms of a dietary deficiency occur• Vitamin B12 is the

precursor of two different cofactor forms that are involved in two very different metabolic reactions

Vit. B12 Metabolism

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• One, catalyzed by methionine synthase (a.k.a. homocysteine methyltransferase), uses methyl-B12 as a one-carbon donor• The other reaction is catalyzed by methylmalonyl-CoA

mutase, which utilizes 5’-deoxyadenosyl-B12 as a cofactor and involves the transfer of a one-carbon unit within the molecule rather than between reactants• This reaction is needed for the conversion of carbons

from valine, isoleucine, threonine, thymine, and the last three carbons of odd-chain fatty acids, all of which form propionyl CoA, to succinyl CoA

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S-Adenosylmethionine• S-Adenosylmethionine (SAM) participates in the

synthesis of many compounds that contain methyl groups• It is used in reactions that add methyl groups to

either oxygen or nitrogen atoms in the acceptor (contrast that to folate derivatives, which can add one-carbon groups to sulfur or to carbon)• More than 35 reactions in humans require methyl

donation from SAM• SAM is synthesized from methionine and ATP; ATP

donates the adenosine and the three phosphates are released • With the transfer of its methyl group , SAM forms

S-adenosylhomocysteine, which is subsequently hydrolyzed to form homocysteine and adenosine• Methionine, required for the synthesis of SAM, is

obtained from the diet or produced from homocysteine, which accepts a methyl group from vitamin B12

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• Thus, the methyl group of methionine is regenerated • The portion of methionine that

is essential in the diet is the homocysteine moiety• If we had an adequate dietary

source of homocysteine, methionine would not be required in the diet• However, there is no good

dietary source of homocysteine, whereas methionine is plentiful in the diet

• Homocysteine provides the S atom for the synthesis of cysteine• In this case, homocysteine reacts with serine to

form cystathionine, which is cleaved, yielding cysteine and α-ketobutyrate• The first reaction in this sequence is inhibited by

cysteine

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• Thus, methionine, via homocysteine, is not used for cysteine synthesis unless the levels of cysteine in the body are lower than required for its metabolic function• An adequate

dietary supply of cysteine, therefore, can “spare” (or reduce) the dietary requirement for methionine

The Synthesis of Cysteine

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The Relationship between Folate, Vitamin B12 and SAM

• In the flow of carbon in the folate cycle, the equilibrium lies in the direction of the N5-methyl FH4 form

• This appears to be the most stable form of carbon attached to the vitamin

• However, in only one reaction can the methyl group be removed from N5-methyl FH4, and that is the methionine synthase reaction, which requires vitamin B12

• The methyl that has been incorporated in methionine is in turn donated to various substrates by SAM

• Thus, if vitamin B12 is deficient, or if the methionine synthase enzyme is defective, N5-methyl FH4 will accumulate

• Eventually most folate forms in the body will become “trapped” in the N5-methy form

• A functional folate deficiency results because the carbons cannot be removed from the folate

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• The appearance of a functional folate deficiency caused by a lack of vitamin B12 is known as the “methyl-trap” hypothesis

Methyl Transfer

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• A dietary deficiency of folate impairs one-carbon metabolism and preferentially impacts rapidly dividing cells, including the stem cells that generate erythrocytes, enterocytes, and cells of the immune system

• The typical clinical presentation of folate deficiency is megaloblastic anemia

• Lack of adequate nucleic acid synthesis results in decreased red cell number and release into the circulation of normochromic red blood cells, which are larger than normal (megaloblasts) due to impaired cell division

• Persons with folate deficiency also often have decreased white cell counts

• Folate deficiency during pregnancy has been associated with an increased risk for neural tube defects in the developing fetus

• There are two major clinical manifestations of vitamin B12 deficiency

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• One such presentation is hematopoietic (caused by the adverse effects of a B12 deficiency on folate metabolism)

• The hematopoietic problems associated with a B12 deficiency (pernicious anemia) are identical to those observed in a folate deficiency and, in fact, result from a folate deficiency secondary to the B12 deficiency

• The underlying problem is a lack of intrinsic factor production by the stomach

• Pernicious anemia is due principally to an autoimmune gastritis in which the blood contains antibodies against intrinsic factor and other proteins of the parietal cells

• These antibodies damage the patient’s mucosa and abolish the secretion of both intrinsic factor and HCl

• The neurologic presentation of vitamin B12 deficiency is though to be caused by hypomethylation in the nervous system and the accumulation of methylmalonyl-CoA

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• When present at elevated concentrations, methylmalonyl-CoA can substitute for malonyl-CoA in fatty acid synthesis, leading to the synthesis of branched-chain fatty acids, which are incorporated into phospholipids of the myelin sheath

o Elevated homocysteine levels have been linked to cardiovascular and neurologic disease. Homocysteine levels can accumulate in a number of ways, which are related to folic acid, vitamin B12, and vitamin B6 metabolism

o Genetic causes of hyperhomocysteinemia include polymorphisms in any of the enzymes involved in either the transsulfuration pathway or the remethylation pathway and related enzymes of folate metabolism

o Even mild hyperhomocysteinemia confers an increased risk of adverse cardiovascular events. Although the mechanism by which homocysteine causes endothelial cell dysfunction is not fully understood, it is thought that homocysteine, which is a potent oxidizing agent, inactivates the vasoprotective agent nitric oxide

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Molecules Derived From Amino AcidsThe Catecholamines • Dopamine and norepinephrine are

neurotransmitters synthesized in the brain• Dopamine, norepinephrine, and epinephrine,

collectively called catecholamines, have two hydroxyl groups on the phenolic ring (catechol is o-dihydroxybenzene)

• The catecholamines are synthesized through a common pathway that starts with tyrosine

• The complete pathway, which generates epinephrine, occurs primarily in the adrenal gland

• The first step in the synthesis of the catecholamines is the hydroxylation of tyrosine by tyrosine hydroxylase, which, like phenylalanine hydroxylase is a mixed-function oxidase that also oxidizes BH4

• Dopamine, is synthesized by decarboxylation of DOPA

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• DOPA decarboxylase, like many other amino acid decarboxylases, utilizes PLP as a cofactor

• Norepinephrine is synthesized by oxidizing dopamine

• Unlike the reactions catalyzed by phenylalanine hydroxylase and tyrosine hydroxylase, dopamine β-hydroxylase oxidizes the side chain rather than the phenyl ring and utilizes ascorbic acid rather than BH4 as the cofactor and reducing agent

• Epinephrine is synthesized by methylating norepinephrine; the methyl donor is SAM

• The two enzymes that inactivate the catecholamines are catechol 0-methyltransferase (COMT) and monoamine oxidase (MAO)

• COMT uses SAM to methylate the hydroxyl group in the 2’-position on the phenyl ring. MAO catalyzes the removal of the terminal amino group of a catecholamine such as dopamine, generating an aldehyde which is then oxidized further to a carboxyl group

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• The reactions catalyzed by COMT and MAO can occur in either order; the resulting degradation products are excreted in the urine

• Parkinson’s disease is associated with low levels of dopamine in the brain

• MAO inhibitors are preferred in the early stages of the disease

• L- Dopa is used for treatment in the late stages

• Dopamine cannot cross the blood- brain barrier; once L-Dopa gets into the brain, it will be changed to dopamine

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Melanin• Melanin is synthesized by specialized cells called

melanocytes, located in the skin, hair roots, and iris and retina of the eye

• Melanocytes contain tyrosinase, a copper-dependent tyrosine hydroxylase that converts tyrosine first to DOPA quinone and then to a family of bicyclic molecules called indoles

• Subsequent oxidation and polymerization of the indoles results in the formation of melanins, whose multiple aromatic rings account for the pigmentation for the skin and hair

• Synthesis of tyrosinase in melanocytes is induced by exposure to UV light

• Lack of melanin production (hypomelanosis) gives rise to several hereditary disorders collectively known as albinism

• Some forms result from deficiency of tyrosinase• Affected individuals have increased susceptibility

to various types of carcinoma from the effect of solar radiation on DNA

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Serotonin and Melatonin• Tryptophan is the precursor of the

neurotransmitter serotonin • Serotonin, in turn, is utilized by the pineal gland to

synthesize melatonin, which regulates seasonal and circadian rhythms

• The pathway for the synthesis of serotonin is similar to that which generates dopamine

• The first step is catalyzed by tryptophan hydroxylase, which, like tyrosine hydroxylase, is a BH4-dependent enzyme

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• DOPA decarboxylase, the enzyme that catalyzes the decarboxylation of DOPA to produce dopamine, then decarboxylates 5-hydroxytryptophan to give serotonin• Serotonin has been implicated in many

processes, including smooth muscle contraction, mood control and appetite regulation• When serotonin levels are low, increased

appetite, or depression, or both can occur• The successive transfer of acetyl and

methyl groups to serotonin yields melatonin• Melatonin is produced in the pineal gland

in response to the light–dark cycle, its level in the blood rising in a dark environment

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• It is probably through melatonin that the pineal gland conveys information about light–dark cycles to the body, organizing seasonal and circadian rhythms

• Melatonin also may be involved in regulating reproductive functions

• One of the alternative pathways for tryptophan catabolism produces niacin, which is the precursor of the nicotinamide component of NAD+ and NADP+

• Niacin synthesis, however, represents a minor pathway for the catabolism of tryptophan; only about 3% of the tryptophan that is metabolized actually follows this pathway

• Monoamine oxidase, which inactivates catecholamines, also catalyzes the oxidative deamination of serotonin to produce 5-hydroxyindole acetic acid

• The activity of a number of antipsychotic drugs is based on inhibiting MAO

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• Cheese and other foods that are processed over long periods (such as red wine) contain tyramine (decarboxylated tyrosine)

• Tyramine induces the release of norepinephrine from storage vesicles, which leads to potentially life-threatening hypertensive episodes

• Usually tyramine is inactivated by MAO-A, but if an individual is taking an MAO inhibitor, tyramine levels will increase –the “cheese effect”

• Selective inhibitors of MAO-A and MAO-B have been produced

Histamine • The decarboxylation of histidine yields histamine• Histamine occurs in blood basophils, tissue mast

cells, certain cells of the gastric mucosa, the pituitary and the brain

• Histamine is involved in such processes as hypersensitivity reactions, HCl secretion, smooth muscle contraction,

• In acute anaphylaxis, bronchiolar constriction is rapidly relieved by epinephrine (a physiological antagonist of histamine)

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• In the brain, histamine is rapidly inactivated by methylation from SAM followed by deamination by MAO which is readily excreted

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γ-Aminobutyric acid (GABA)• The excitatory neurotransmitter glutamate is

decarboxylated to produce the inhibitory GABA

Nitric Oxide • NO is unique among the

neurotransmitters in that it is lipophilic and can diffuse rapidly across biological membranes

• NO mediates a variety of physiological functions such as endothelial derived relaxation of vascular smooth muscle, inhibition of platelet aggregation, neurotransmission, and cytotoxicity

• NO is synthesized from one of the terminal nitrogen atoms or the guanidino group of arginine with the concomitant production of citrulline

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• Molecular oxygen and NADPH are cosubstrates and the reaction is catalyzed by nitric oxide synthase (NOS)

• NOS is a complex enzyme containing bound FMN, FAD, BH4, heme complex, and non-heme iron. A calmodulin binding site is also present

• Citrulline can be recycled back to arginine by the enzymes of the urea cycle

• There are three isozymes of NO synthase. The neural (nNOS) and endothelial (eNOS) forms are constitutive isoforms regulated by the intracellular calcium concentration and produce NO for its neurotransmitter and local hormone roles

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• By contrast, the isozyme induced in activated macrophages (iNOS) produces NO that contributes to the bacteriocidal response; iNOS is calcium-independent

• In the vascular endothelium, agonists such as acetylcholine and bradykinin activate eNOS by enhancing intracellular Ca 2+ concentrations via the production of IP3

• The NO produced in the vascular endothelium maintains basal vascular tone by vasodilation which is mediated by vascular smooth muscle cells

• Organic nitrates used in the management of ischemic heart disease act by denitration with the subsequent formation of NO

Creatine • Synthesis of creatine (methyl guanidinoacetate)

requires the transfer of a guanidine group from arginine to glycine, to form guanidinoacetate ; this occurs in the kidney

• In the next step guanidinoacetate is methylated by SAM in the liver

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The Metabolism of Creatine

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• The creatine formed is released from the liver and travels through the bloodstream to other tissues, particularly skeletal muscle, heart, and brain, where it reacts with ATP to form the high-energy compound creatine phosphate

• This reaction, catalyzed by creatine kinase (CK) is reversible. Therefore, cells can use creatine phosphate to regenerate ATP

• Creatine phosphate is an unstable compound. It spontaneously cyclizes, forming creatinine

• Creatinine cannot be further metabolized and is excreted in the urine

• The daily excretion of creatinine depends on skeletal muscle mass and varies with age and sex

• Creatinine clearance approximately parallels the glomerular filtration rate (GFR) and is used as a kidney function test

• Creatinuria, the excessive excretion of creatine in urine, may occur during growth, fever, starvation, diabetes mellitus, extensive tissue destruction,…

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Polyamines• Polyamines have multiple positive charges that

stabilize DNA during cell division and are therefore essential for cell survival

• Polyamines are present in all cells in relatively high, often millimolar, concentrations

• Putrescine, the simplest of the polyamines, is produced by decarboxylation of ornithine

• The larger, more positively charged polyamines, spermidine and spermine, are synthesized by means of the transfer of aminopropyl groups to putrescine

• In this pathway, SAM is first decarboxylated• Transfer of an aminopropyl group from

decarboxylated SAM to putrescine generates spermidine; transfer of a second aminopropyl group to spermidine generates spermine

• The decarboxylation of lysine and arginine would lead to cadaverine and agmatine , respectively

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The Structure and

Synthesis of Polyamines

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The Metabolism of HemeHeme Biosynthesis• The principal tissues involved in heme biosynthesis

are the hematopoietic tissues and the liver• Biosynthesis requires participation of eight

enzymes, of which four (the first and the last three) are mitochondrial and the rest are cytosolic

• The reactions are irreversible• Heme is synthesized from glycine and succinyl

CoA , which condense in the initial reaction to form δ-aminolevulinic acid (δ-ALA)

• The enzyme that catalyzes this reaction, δ-ALA synthase, requires the participation of PLP, as the reaction is an amino acid decarboxylation reaction (glycine is decarboxylated)

• The next reaction of heme synthesis is catalyzed by δ-ALA dehydratase, in which two molecules of δ-ALA condense to form the pyrrole, porphobilinogen

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• Four of these pyrrole rings condense to form a linear chain and then a series of porphyrinogens

• The side chains of these porphyrinogens initially contain acetyl (A) and propionyl (P) groups

• The acetyl groups are decarboxylated to form methyl (M) groups

• Then the first two propionyl side chains are decarboxylated and oxidized to vinyl (V) groups, forming a protoporphyrinogen

• The methylene bridges are subsequently oxidized to form protoporphyrin IX

• In the final step of the pathway, ferrous iron is incorporated into protoporphyrin IX in a reaction catalyzed by ferrochelatase (also known as heme synthase)

• To produce one molecule of heme, 8 molecules each of glycine and succinyl CoA are required

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The Synthesis of Heme and Associated Disorders

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Iron Metabolism• The average man and woman contain about 3500

and 2600 mg of iron, respectively• The hemoglobin of red blood cells and the

myoglobin of muscle cells account for most of this iron

• Approximately 0.8% of a person's red blood cells are broken down each day by the reticuloendothelial system, which results in the release of 20 mg of iron into the blood

• Ninety-five percent of this iron is recycled and reutilized by the bone marrow to synthesize new red blood cells, which replace those that were broken down

• Iron, which is obtained from the diet, has a Recommended Dietary Allowance (RDA) of 10 mg for men and postmenopausal women, and 15 mg for premenopausal women

• The iron in meats is in the form of heme, which is readily absorbed

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• The non-heme iron in plants is not as readily absorbed in part because plants often contain compounds that chelate or form insoluble precipitates with iron, preventing its absorption.

• Conversely, vitamin C increases the uptake of non-heme iron from the digestive tract

• Iron is absorbed in the ferrous (Fe2+) state, but is oxidized to the ferric state by a ferroxidase known as ceruloplasmin (a copper-containing enzyme) for transport through the body

• Because free iron is toxic, it is usually found in the body bound to proteins

• Iron is carried in the blood (as Fe3+) by the protein apotransferrin, with which it forms a complex known as transferrin

• Transferrin is usually only one-third saturated with iron

• Storage of iron occurs in most cells but especially those of the liver, spleen, and bone marrow

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• In these cells, the storage protein, apoferritin, forms a complex with iron (Fe3+) known as ferritin

• Normally, little ferritin is present in the blood. This amount increases, however, as iron stores increase

• Iron can be drawn from ferritin stores, transported in the blood as transferrin, and taken up via receptor-mediated endocytosis by cells that require iron (e.g., by reticulocytes that are synthesizing hemoglobin)

• When excess iron is absorbed from the diet, it is stored as hemosiderin, a form of ferritin complexed with additional iron that cannot be readily mobilized

• About 1 mg of iron is lost each day through exfoliation of skin and intestinal cells

• This iron loss is made up for by the absorption of an equivalent amount of iron from the intestine

• Only round 10% of the dietary iron is absorbed

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Iron Metabolism

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Regulation of Heme Synthesis• δ -ALA synthase catalyzes the regulated step of

heme synthesis• Both the synthesis and activity of the enzyme are

inhibited by heme and by hemin (which contains ferric iron)

• There are two isoforms of δ-ALA synthase: δ-ALA S 1 in non-erythroid cells and δ-ALAS2 in erythroid cells and their expression is regulated differently

• Essentially all of the heme made by erythroid cells is committed to hemoglobin synthesis

• Hypoxia and erythropoietin increase heme synthesis in erythroid cells

• δ -ALAS2 mRNA contains an iron-responsive element (IRE) and is responsive to the intracellular availability of iron

• Heme synthesis is also coordinated with globin-chain protein synthesis

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• By contrast, most of the heme synthesized in hepatocytes is incorporated into cytochromes of the electron-transport chain and P450-type cytochromes involved in biotransformation

• The expression of δ-ALAS1 in hepatocytes is increased in response to many of the drugs and toxins that are metabolized in the liver

Degradation of Heme• Heme is degraded to form bilirubin, which is

conjugated with glucuronic acid and excreted in the bile

• Although heme from cytochromes and myoglobin also undergoes conversion to bilirubin, the major source of this bile pigment is hemoglobin

• After red blood cells reach the end of their lifespan, they are phagocytosed by cells of the reticuloendothelial system

• Globin is cleaved to its constituent amino acids, and iron is returned to the body’s iron stores

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• The conversion of heme to bilirubin can be visualized in a bruise that is initially reddish purple (heme) and with time turns yellow-green (biliverdin) and then red-orange (bilirubin)

• The initial reaction that cleaves the porphyrin ring is catalyzed by heme oxygenase, producing biliverdin IX and carbon monoxide, and concurrently releasing the oxidized Fe3+ ion:

Heme+3O2+3NADPH+3H+→Biliverdin + CO + Fe3+

+3NADP+ +3H2O• Biliverdin reductase then reduces biliverdin, to give

bilirubin • Although all cells contain heme oxygenase and can

convert heme generated during turnover of hemoproteins to bilirubin, only the liver is capable of converting bilirubin to the more water-soluble bilirubin diglucuronide

• Bilirubin is first transported to the liver complexed with serum albumin

• UDP-glucuronyl transferase then catalyzes the successive transfers of two glucuronic acid residues from UDP-glucuronic acid to bilirubin

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The Synthesis and Conjugation of Bilirubin

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• Conjugation with glucuronic acid is also the mechanism the liver uses to increase the solubility of steroids and certain drugs prior to their excretion

• Clinically, conjugated bilirubin or bilirubin diglucuronide is often called direct-acting bilirubin and unconjugated bilirubin is called indirect-acting bilirubin

• This nomenclature is related to the colorimetric reaction which is commonly used to quantify the two forms of bilirubin which is important for the differential diagnosis of the causes of hyperbilirubinemia

• In this assay, conjugated bilirubin reacts readily with an azo dye

• Unconjugated bilirubin, on the other hand, is much more lipophilic and tightly bound to serum albumin; it must be released with alcohol before the dye-coupling reaction can occur

• The assay first quantifies conjugated bilirubin; then, with the addition of alcohol, the test quantifies total plasma bilirubin

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• The quantity of unconjugated bilirubin is determined by subtraction, and unconjugated bilirubin is therefore designated indirect bilirubin

• Conjugated bilirubin is released into the bile (giving it its color) and delivered into the small intestine when the gallbladder contracts

• In the lower intestine and colon, bacterial β-glucuronidases remove glucuronic acid to form unconjugated bilirubin

• Further metabolism of bilirubin by bacteria reduces bilirubin to a colorless tetrapyrrolic compound called urobilinogen

• A small amount of urobilinogen is absorbed and enters into the enterohepatic circulation; a minor fraction of this urobilinogen is ultimately excreted by the kidney, partly as the oxidized, colored compound urobilin, which imparts the characteristic yellow color of urine

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• Most of the urobilinogen formed in the gut is further metabolized by the enteric bacteria to stercobilinogen and excreted mainly in its oxidized form, stercobilin, which imparts the characteristic color of stool

Diseases Involving Heme and Iron MetabolismIron Deficiency Anemia • Iron deficiency can result from inadequate intake

of iron or loss of iron resulting from hemorrhage (e.g., gastrointestinal-tract bleeding) or excessive menstrual blood loss

• Globally, inadequate dietary intake of iron remains the major cause of iron deficiency, especially where the diet is largely cereal-based and contains little meat

• Iron-deficiency anemia is a major cause of pregnancy-related mortality in developing countries

• In iron-deficiency anemia, red blood cells are small (microcytic) and pale (hypochromic)

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• Prior to development of frank anemia, the most sensitive clinical indicator of emerging iron deficiency is a plasma ferritin concentration that falls below the reference range

• When iron stores become so low as to compromise erythropoiesis, there is an increase in the serum concentration of transferrin (increased iron binding capacity) and a decrease in transferrin saturation (less than the normal 30% of the iron-binding sites of transferrin occupied by iron atoms)

• Iron deficiency is commonly treated by supplementing the diet with ferrous salts (e.g., ferrous sulfate) and juices containing ascorbic acid and citric acid which enhance iron absorption

Hemosiderosis• Hemosiderin accumulates in macrophage due to an

increased phagocytosis of red blood cells associated with hemorrhage

• Accumulation of hemosiderin in liver is associated with iron overload, as can occur in people who receive frequent blood-transfusion for sickle cell anemia or thalassemia

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Lead Poisoning• Lead inhibits both δ-ALA dehydratase and

ferrochelatase, thereby reducing heme synthesis and resulting in microcytic, hypochromic anemia

• Plasma δ-ALA and erythrocyte protoporphyrin concentrations are increased in people with lead poisoning

• Furthermore, since heme is the prosthetic group of many enzymes and proteins, including the cytochromes of the mitochondrial electron-transport chain, lead poisoning can also have detrimental effects on energy metabolism

• Lead is especially toxic to the nervous system, probably due to accumulation of δ-ALA as well as to impaired energy metabolism

Porphyrias • Porphyrias are a group of rare inherited disorders

resulting from deficiencies of enzymes in the pathway for heme biosynthesis

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• Depending on the particular gene affected, porphyrias can affect heme synthesis in all cells or be primarily either hepatic or erythropoietic

• The nervous system is also usually affected leading to neuro-psychiatric symptoms

• When porphyrinogens accumulate, they may be converted by light to porphyrins, which react with molecular oxygen to form oxygen radicals

• These radicals may cause severe damage to the skin. Thus, individuals with excessive production of porphyrins are photosensitive

Jaundice• Jaundice (also known as icterus) is a condition of

impaired heme catabolism • It is characterized by a yellow color of the skin and

sclerae of the eyes that is the result of an elevated plasma concentration of bilirubin

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•Bilirubin toxicity or kernicterus occurs when the plasma level of bilirubin is high enough to result in transfer of excess bilirubin to membrane lipids, particularly in the brain• Jaundice can be a symptom of many different clinical problems

Pre-hepatic Jaundice• In hemolytic anemias, the excess breakdown of RBC results in the production of abnormally large quantities of bilirubin, which may overload the liver’s capacity to conjugate bilirubin•As a result, the plasma concentration of unconjugated bilirubin rises•Unconjugated bilirubin may also spill over into bile and increase the risk of developing pigmented gallstones (calcium bilirubinate)

Hepatic Jaundice • Impaired liver function is one of the major causes of jaundice • Hepatitis and cirrhosis impair the ability of hepatic UDP-glucuronyl transferase to conjugate bilirubin

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• The secretion of conjugated bilirubin into the bile is also compromised

• As a result, both unconjugated and conjugated bilirubin accumulate in the blood, while fecal and urinary urobilinogen levels are decreased

• Hepatic jaundice can also result from deficiency of one or more of the enzymes involved in the metabolism and excretion of bilirubin

Post-hepatic Jaundice• Obstruction of the common bile duct due to a stone

or (less commonly) a tumor results in post-hepatic jaundice

• The backup of conjugated bilirubin in the liver results in abnormal spillage of conjugated bilirubin into the blood and its excretion in the urine, thereby imparting a dark color

• By contrast, lack of biliary excretion results in pale stools that lack normal pigmentation

• Enzyme defects could also lead to post-hepatic jaundice

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Neonatal Jaundice• Many (60%) full-term newborns develop neonatal

jaundice• This is usually caused by an increased destruction

of red blood cells after birth (the fetus has an unusually large number of red blood cells) and an immature bilirubin conjugating system in the liver

• This leads to elevated levels of non-conjugated bilirubin, which is deposited in hydrophobic (fat) environments

• If bilirubin levels reach a certain threshold at the age of 48 hours, the newborn is a candidate for phototherapy, in which the child is placed under lamps that emit light between the wavelengths of 425 and 475 nm

• Bilirubin absorbs this light, undergoes chemical changes, and becomes more water soluble. The products can be excreted by way of the liver without requiring glucuronic acid conjugation

• Usually, within a week of birth, the newborn’s liver can handle the load generated from red blood cell turnover