Download Amino Acid Metabolism

Amino acid metabolism
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
•
•
•
•
•
•
•
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
2
increased under conditions of fasting and starvation
• Plants and some bacteria synthesize all 20 amino acids.
Humans (and other animals) can synthesize some (the nonessential 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
3
• 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
4
and feces) exceeds that taken in
• 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 repair
2. Synthesis of a range of nitrogen-containing small compounds
3. 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
5
energy production. The ammonia is converted to urea
•
•
•
•
•
•
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
6
protease pepsin
• 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
7
the secretions from the exocrine pancreas
• 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. Intracellular peptidases act on small peptides that are absorbed 8
9
The Specificity of Proteases
•
•
•
•
•
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
10
serosal membrane
• 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
11
•
•
•
•
•
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
12
sufficient protein to sustain healthy growth
• 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
13
retardation
•
•
•
•
•
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
14
2. The regulation of hormone and enzyme levels
• There are two main pathways for protein degradation:
the lysosomal-autophagic system and the ubiquitinproteasome 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 longlived intracellular proteins are degraded in lysosomes by
ATP-independent processes
15
• 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
16
Lysosomal Degradation
17
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,
18
is a small (8.5 kDa, 76 aa residues) protein
• 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
19
sequences
• 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
20
• 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
21
join the intracellular pool of free amino acids
The Proteasome
22
• 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
23
• 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
24
•
•
•
•
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
25
• 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
26
α- ketoglutarate, forming glutamate
Fed State
Post-absorptive State
27
• 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-ketoglutarate, 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
28
PLP
The Mechanism of
Transamination Reactions
29
• 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
30
• 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
31
• 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
32
• 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
33
forms of nitrogen in the blood
• 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
34
into the urine as a buffer
• 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 NH354+
• Bacteria in the gut also act on amino acids, urea and other
nitrogen containing molecules to release free ammonia
36
•
•
•
•
•
•
•
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
37
than urea, including uric acid, creatinine, and ammonia
• 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 Cycle
Synthesis 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
38
required to form the high-energy phosphate bond of CP
• 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
39
occurs in mitochondria
• 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
40
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
41
• 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
42
• 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
43
The Urea Cycle
44
• 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 enzymes45
• 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
46
urea cycle enzymes
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
47
hepatic blood flow
• 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 48ATP
• 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
49
• 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 ureacycle 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
50
arginine supplementation is beneficial
• 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
51
excretion
•
•
•
•
•
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;
52
lysine and leucine are strictly ketogenic
53
• 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
54
overlap (e.g. pyruvate, oxaloacetate, α-ketoglutarate)
The Precursors for
Non-Essential Amino Acids
55
Amino Acid Metabolism and Abnormalities
Glycine
• 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
56
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
57
over” into the urine
• 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
58
also the major pathway for the catabolism of excess phenylala.
Branched-Chain
Amino Acid
Metabolism
59
• 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
60
The Regeneration
of BH4
• 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,
61
phenylacetate, and other metabolites of phenylalanine
• 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
62
• 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 63
One-Carbon Metabolism
• 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)
64
• 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
65
generation of thymidylate for DNA synthesis
• 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
66
successive reduction steps
• 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
5
10
5
10
nitrogen N or N or they form a bridge between N and N
5
• Groups that can be attached to N include formyl (-CHO),
formimino (-CH=NH), or methyl (-CH3 ) groups
10
• N can carry formyl groups
• Methylene (-CH2- ) or methenyl (-CH=) groups form bridges
5
10
between N and N
67
• 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 onecarbon pool that was donated
at a different oxidation state
• Once the methyl group is
formed, it is not readily
5
10
reoxidized back to N , N
5
methylene FH4, and thus N methyl-FH4 will tend to
68
accumulate in the cell
The One-Carbon Pool
• Carbon sources for the onecarbon 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
5
10
and N , N -methylene- FH4
• Because serine can be synthesized
from 3-phosphoglycerate, dietary
carbohydrate can serve as a
source of carbon for the one69
carbon pool
• 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
5
N -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
70
the combined hydrolytic actions of HCl and pepsin
• 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
71
and then is released into circulation
• 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
72
• 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 oddchain fatty acids, all of which form propionyl CoA, to succinyl CoA
73
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
74
methyl group from vitamin B12
• 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 75
• 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
76
•
•
•
•
•
•
•
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
77
cannot be removed from the folate
• The appearance of a functional folate deficiency caused by a
lack of vitamin B12 is known as the “methyl-trap” hypothesis
Methyl Transfer
78
• 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
79
deficiency
• 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
80
system and the accumulation of methylmalonyl-CoA
• 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
81
oxidizing agent, inactivates the vasoprotective agent nitric oxide
Molecules Derived From Amino Acids
The 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 82
83
• 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
84
carboxyl group
The Inactivation of Cathecolamines
• 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 LDopa gets into the brain, it
will be changed to dopamine
85
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
86
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
87
• 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
88
89
• 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
90
• 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) 91
The Synthesis and Inactivation of
Histamine
• In the brain, histamine is rapidly inactivated by methylation
from SAM followed by deamination by MAO which is readily
excreted
92
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
93
citrulline
• 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
94
produce NO for its neurotransmitter and local hormone roles
• 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
95
liver
The Metabolism of Creatine
96
• 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,
97
extensive tissue destruction,…
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
98
99
The Metabolism of Heme
Heme 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
100
form the pyrrole, porphobilinogen
• 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
101
The Synthesis of Heme and
Associated Disorders
102
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
103
• 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
104
• 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
105
Iron Metabolism
106
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 nonerythroid 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
107
synthesis
• 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
108
• 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
109
acid to bilirubin
The Synthesis and
Conjugation of Bilirubin
110
• 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 bilirubin111
• 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
112
characteristic yellow color of urine
• 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 Metabolism
Iron Deficiency Anemia
• Iron deficiency can result from inadequate intake of iron or
loss of iron resulting from hemorrhage (e.g., gastrointestinaltract 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)
113
• 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 ironbinding 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 blood114
transfusion for sickle cell anemia or thalassemia
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
115
• 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 neuropsychiatric 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
116
of bilirubin
• 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 UDP117
glucuronyl transferase to conjugate bilirubin
• 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 118
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
119
the load generated from red blood cell turnover