Amino Acid Metabolism LE6

DR. AGNES ALARILLA-ALBA, MD, FPPS,FPSN,FPNSP MM/DD/YYYY

credentials)
OUTLINE Biomedical Importance
I. Overview of Amino
V. Protein Catabolism  Amino acids deficiency will result if the essential amino acids
Acids are absent from the diet or present in an inadequate amount.
VI. Urea Cycle
II. Classification of Amino
VII. Metabolic Disorders of  Kwashiorkor which results when a child is on a starchy diet poor
Acids in protein. (Low protein but enough calories)
Urea Cycle
III. Biosynthesis of
VIII. Regulation of Urea  Patients with short bowel syndrome is unable to absorb
Nutritionally sufficient quantities of calories and nutrients including protein.
Cycle
Nonessential Amino
IX. Case
Acids
X. Review Questions
IV. Amino Acids Derived
XI. References
from Intermediates of
XII. Appendix
Processes

LEARNING OBJECTIVES

At the end of the lecture, the student should be able to:

1. To be able to classify the nutritionally essential from the
nutritionally nonessential amino acid
2. To be able to determine the key role of the
transaminases in amino acid metabolism
3. To determine the intermediates of the citric acid cycle
and glycolysis that are precursors of some amino acid

I. Overview of Amino Acids

 Amino acids are the basic building blocks of the body. They are
also sources of energy, like fats and carbohydrates. However,
amino acids are structurally characterised by the fact that they Figure 2. Short bowel Syndrome
contain nitrogen (N), compared to fats and carbohydrtaes.
 Amino acids are extremely versatile. Although more than 200 B. Metabolic Interrelationships of Amino Acids
different amino acids exist
 In the form of proteins, amino acid residues form the second-
largest component (water is the largest) of human muscles and
other tissues

A. Uses of Amino Acids

Metabolic Uses
 Substrates for protein synthesis and other nitrogen containing
compound
 Subsrates for the synthesis of other products like heme, purine,
pyrimidine and others (hormones, nuerotransmitters
 Used as substrates for generation of metabolic energy Figure 2. Metabolism of Amino Acids

Major Site of Metabolism

 Liver is the major site of amino acid metabolism in the body and
the major site of synthesis (Marks, 4th.ed)
 Liver is also the major site of amino acid degradation
 Hepatocytes partially oxidize most amino acids, converting the
carbon skeleton to glucose, ketones and CO2
 Since ammonia is toxic to our body, liver converts most of the
nitrogen from amino acid degradation to urea which is excreted
in urine.

Protein Turnover
 Protein turnover is the net result of continuous synthesis and
breakdown of body proteins and ensures maintenance of
optimally functioning protein
 The amino acid pool describes the entire amount of available
free amino acids in the human body. The size of the pool
amounts to around 120 to 130 grams in an adult male.
Figure 1. Uses of Amino Acids

Trans # Group #24 : Damasco, Damasco, Danganan, Daquial EDITOR: Cruz 1 of

17
TiTa
Amino Acid Metabolism 1
C. Metabolism of Dietary and Endogenous Protein II. CLASSIFICATIONS OF AMINO ACIDS

A. BASED ON STRUCTURE

Based on R-chain structure

Table 1. Amino Acids Based on R-Chain

Classes of Amino Acids Name of the Amino Acid

Glycine, Alanine, Valine,
Aliphatic
Leucine, Isoleucine
Serine, Cysteine, Threonine,
Hydroxyl or Sulfur-containing
Methionine
Cyclic Proline
Phenylalanine, Tyrosine,
Aromatic
Tryptophan

Based on Polarity an Charge (Review)

Figure 3. Metabolism of Dietary Proteins

Dietary Proteins (Review Only)

 Most of the ingested protein is ultimately oxidised to provide
energy, and the surplus nitrogen is excreted, a little as
ammonia but mostly as urea.
 Dietary proteins are initially denatured by the stomach acid, in
conjunction with limited proteolysis by pepsin
 Protein digestion is largely completed in the small intestine at a
slighlty alkaline pH. The pancreatic proteases trypsin,
Figure 4. Amino acids based on polarity and charge (Note: Tyrosine can
chymotrypsin and elastase divide the proteins into short
also be classified to polar, uncharged)
peptides. These are attacked from both ends by
aminopeptidase and carboxypeptidase, and the fragments are
finished off by dipeptidases secreted from the gut wall. B. BASED ON NATURE OF SYNTHESIS AND
 Amino acid uptake from the gut lumen into enterocytes is HUMAN NUTRITION
driven by the sodium gradient. There is a relatively high sodium
concentration in the gut (regardless of dietary intake, as a Proteinogenic vs Non-proteinogenic
result of the pancreatic secretion of sodium bicarbonate) and a  Proteinogenic amino acids are broadly defined as the amino
low concentration in the enterocytes, as a result of the sodium acids that are used to form proteins.
pump in the basolateral membrane. A multiplicity of sodium-  Subjected to translation attached to tRNA used to form the
linked amino acid carriers operate within the intestinal brush primary polypeptide structure of proteins
border, balanced by sodium-independent export carriers on the  There are 22 proteinogenic ("protein-building") amino acids,
serosal surface (i.e. the opposite side) of the cells  These are all L-stereoisomers, although a few D-amino acids
occur in bacterial envelopes, as a neuromodulator (D-serine),
Endogenous Proteins and in some antibiotics.
 A typical muscle protein might survive for three weeks, but  Non-proteinogenic amino acids are not required to build
many liver enzymes turn over in a couple of days. Some proteins (almost 700 of these). However, this doesn’t mean that
regulatory enzymes have half-lives measured in hours or they are not important. These amino acids have a vital role as
minutes. The majority of the amino acids released during metabolic intermediate (eg: carnitine, creatine)
protein degradation are promptly re-incorporated into fresh
proteins. Net protein synthesis accounts for less than one third Standard vs Non-standard
of the dietary amino acid intake, even in rapidly growing children  Twenty of the proteinogenic amino acids are encoded
consuming a minimal diet. directly by triplet codons in the genetic code and are known as
 During Protein Turonver: standard amino acids. These that are formed from universal
 Soluble intracellular proteins are tagged for destruction by genetic coding. Standard amino acids (also known as canonical
attaching ubiquitin. amino acids) are protein ‘building-blocks’. These are the amino
 They are then degraded in proteasomes to short peptides. acids that together with DNA help organism to form and
They are further metabolized to free amino acids. Some function.
proteins are degraded by an alternative system within the  The other two non-standard are selenocysteine, and pyrrolysine
lysosomes  Synthesized by exploiting genetic code that has not been
 During fasting and starvation muscle protein and adipose used or in a certain position within an amino acid chain.
lipids are consumed to support gluconeogenesis  Non-standard amino acids can also be modified forms of
 Proteinssubstantial ENERGY source standard amino acids.
BIOCHEMISTRY 2 of
17
TiTa
Amino Acid Metabolism 1
Nutritionally Essential vs Non-essential  Arginine/Histidine -- conditionally essential must be
 A balanced diet usually ensures that the body acquires enough provided in the diet when the body’s ability to synthesize them is
essential amino acids outstripped, such as during periods of rapid growth (as in
 Acids -cannot be synthesized by the body; must be present in infancy & childhood) or during recovery from illness.
the diet  Methionine – required in large amounts to produce cysteine if
 PVT TIM HALL the latter is not supplied adequately by the diet.
 Always (Arg)ues, Never (Tyr)s  Phenylalanine - needed in larger amounts to form tyrosine if
 The “A” is always Arg = Arginine the latter is not supplied adequately by the diet
 The 2 “T’s” are Threonine and Tryptophan but never Tyr
= Tyrosine C. BASED ON METABOLIC USE
 Nonessential Amino acids -can be synthesized by the body
 Arginine- Nutritionally “semi-essential”, synthesized at rates Figure 5. Glucogenic vs Ketogenic AAs
inadequate to support growth of children Glucogenic Glucogenic Ketogenic
 Hydroxyproline and Hydroxylysine- not necessary for protein and
synthesis, but is formed during post translational processing of Ketogenic
collagen Non-essential Alanine Tyrosine
 All AA are essential to health Asparagine
 Of the 20 AA: Aspartate
 8 must be present in human diet Cysteine
 12 are nutritionally non essential because they don’t need to Glutamate
be present in the diet Glutamine
 Dependence on an external supply can be of greater survival Glycine
value than the ability to biosynthesize it Proline
Serine
Figure 4. Essential vs Non-essential AAs Essential Arginine Isoleucine Leucine
Nutritionally Essential Nutritionally Non Histidine Phenylalanine Lysine
Essential Methionine Tryptophan
 Phenylaline  Alanine Valine Threonine
 Valine  Aspargine
 Tryptophan  Aspartate III. BIOSYNTHESIS OF NUTRITIONALLY
 Threonine  Cysteine NONESSENTIONAL AMINO ACIDS
 Isoleucine  Hydroxyproline
 Methaionine  Glutamate A. Overview of the Nutritionally Essential Amino
 Histidine  Glutamine
Acids
 Arginine  Glycine
 Leucine  Hydroxyproline
 Lysine  Proline
 Serine
 Tyrosine

Figure 6. Overview of synthesis of the nonessential amino acids

Importance
 Synthesis of AA is essential for:
 Critical for cell survival
 Building blocks for proteins but also as starting points for the
synthesis of many important cellular molecules including
vitamins and nucleotides
 Units reflect either common mechanisms or the use of
 Essential Amino Acids has LARGER enzyme requirement for
common enzymes that synthesize more than one amino
synthesis compared to Non-Essential Amino Acids
acid.
 The number of enzymes required to synthesize essential amino
 These categories are: simple reactions, branch chain
acids is large relative to the number of enzymes required to
amino acids, aromatic amino acids, threonine/lysine,
synthesize the nonessential amino acids (Table 2) → suggests
serine/glycine, and unique pathways.
a survival advantage in retaining the ability to manufacture
 The aromatic amino acids, threonine/lysine and
“easy” amino acids while losing the ability to make “difficult”
serine/glycine pathways have a common beginning and
amino acids
then diverge to form the amino acid of interest.
BIOCHEMISTRY 3 of
17
TiTa
Amino Acid Metabolism 1
 Each pathway begins with a central metabolite C. Glutamine
 Using common compounds instead of synthesizing them from
scratch saves energy and conserves genes since fewer  Amidation of glutamate to glutamine catalyzed by glutamine
enzymes are needed to code for the pathways synthetase involves the intermediate formation of
 ƴ glutamyl phosphate and ADP
 NH4 then binds and as NH3 attacks the glutamylphosphate to
Intermediate Sources of Amino Acids
form a tetrahedral structure release of P1 and a proton releases
 Note: Some non-essential amino acids came from essential the product glutamine
AAs  Can be converted to glutamate by glutamine aminohydrolase
 The α-ketoglutarate family: synthesis of glutamate, glutamine,
proline and arginine (not applicable in humans)
 The oxaloacetate/aspartate family of amino acids is composed
of lysine, asparagine, methionine, threonine, and isoleucine.
 Aspartate can be converted into lysine, asparagine,
methionine and threonine.
 Threonine also gives rise to isoleucine.
 3-phosphoglycerate family: Serine, Glycine and Cysteine
 Reactions beginning with either one or two molecules of
pyruvate cause the synthesis of alanine, valine, and leucine.
 Phenylalanine (not in humans) tyrosine, and tryptophan
(not in humans) are known as the aromatic amino acids. The
synthesis of all three shares a common beginning to their
pathways; the formation of chorismate from
phosphoenolpyruvate (PEP) and erythrose 4- phosphate (E4P).
 Hydroxylation of Phenylalanine yields Tyrosine

SIMPLE REACTIONS: Glutamine, Glutamate,

Aspartate, Asparagine, Alanine Figure 8. Overview of synthesis of Glutamine

 Can be synthesized by one step reactions from central NOTES:

metabolites. GLUTAMATE DEHYDROGENASE, GLUTAMINE
 simple in structure SYNTHETASE, AMINOTRANSFERASES
 synthesis is also straight forward. THEY play a central role in amino acid biosynthesis
 Glutamate can by synthesized by the addition of ammonia to a- Converts inorganic ammonium ion into the alpha amino
ketoglutarate nitrogen amino acids

B. Glutamate
D. Alanine and Aspartate

Alanine transaminase (ALT)

 Alanine aminotransferase (ALAT) or SGPT (serum glutamic
pyruvic transaminase)
 ALT catalyzes the transfer of an amino group from L-alanine to
α-ketoglutarate, vice versa.
 Transamination of PYRUVATE forms ALANINE
 Amino Donor: Glutamate (or aspartate)
 Other product: a ketoglutarate (or oxaloacetate)

Aspartate transaminase (ALT)

 aspartate aminotransferase, also known as AspAT/ASAT/AAT
or serum glutamic oxaloacetic transaminase (SGOT)
 Transamination of OXALOACETATE forms ASPARTATE via
 PLP-dependent
 Amino Donor: glutamate
 Other product: a ketoglutarate
Figure 7. Overview of synthesis of Glutamate
 First reaction in the biosynthesis of “glutamate family”
 REDUCTIVE AMIDATION of alpha ketoglutarate catalyzed by
mitochondrial GLUTAMATE DEHYDROGENASE
 Physiologically important that reaction favors glutamate
because high concentration of AMMONIUM is cytotoxic
 Glutamate Dehydrogenase- removes amino group from the
carbon skeleton of glutamate IN THE LIVER

Glutamate
 Free ammonia was released by oxidative deamination of
Glutamate
Figure 9. Overview of synthesis of Alanine

BIOCHEMISTRY 4 of
17
TiTa
Amino Acid Metabolism 1

Figure 12. Overview of synthesis of Serine

F. Glycine

 Glycine aminotransferase can catalyze synthesis of GLYCINE

FROM glyoxylate AND glutamate OR Alanine
 Other source of glycine is from serine and choline
 Strongly favor glycine synthesis

Figure 10. Overview of synthesis of Aspartate and Alanine

Formation of Glycine from Choline
 Choline is converted to Betaine Aldehyde by CHOLINE
D. Asparagine DEHYDROGENASE
 Betaine Aldehyde is then converted to Betaine by BETAINE
DEHYDROGENASE
 Conversion of aspartate to asparagine catalyzed by  Betaine is converted to Dimethylglycine by BETAINE
ASPARAGINE SYNTHETASE HOMOCYSTEINE N-METHYLTRANSFERASE
 Resembles glutamine synthetase reaction, but GLUTAMINE,  Dimethylglycine is converted to Sarcosine by SARCOSINE
rather than ammonium ion, provides the nitrogen DEMETHYLASE
 Reaction involves the intermediate formation of aspartyl  Finally, Sarcosine is converted to Glycine by SARCOSINE
phosphate OXIDASE
 Since GLUTAMINE provided the nitrogen it will become
GLUTAMATE, thus the product of the reaction are
ASPARAGINE AND GLUTAMATE

Figure 11. Overview of synthesis of Asparagine

E. Serine

 Steps:
 Oxidation of the alpha hydroxyl group of the glycolytic
intermediate 3-phospoglycerate by 3 Figure 13. Overview of synthesis of Glycine from Choline
PHOSPOGLYCERATE DEHYDROGENASE converts it to
3 phosphohydroxypyruvate. Formation of Glycine from Serine
 Transamination
 Glycine can be converted to serine by reversible addition of a
 Subsequent dephosphorylation
methylene group from N5,N10- methylenetetrahydrofolic acid
 Formation of SERINE
 Occurs at LIVER AND KIDNEY

BIOCHEMISTRY 5 of
17
TiTa
Amino Acid Metabolism 1

Figure 13. Interconversion of Serine and Glycine

G. Proline

 Proline is the only proteinogenic amino acid with a secondary

amine, in that the alpha-amino group is attached directly to the
side chain, making the α carbon a direct substituent of the side Figure 15. Formation of Cysteine
chain.
 Proline formation is formed from glutamate by reversal of the I. Tyrosine
reactions of proline catabolism.
 The initial reaction of proline biosynthesis converts the γ-
carboxyl group of glutamate to the mixed acid anhydride of  In plants and most microorganisms, Prephenate is oxidatively
glutamate γ-phosphate. Glutamate-5-semialdehyde is first decarboxylated with retention of the hydroxyl group to give p-
formed by glutamate 5-kinase (ATP-dependent) and glutamate- hydroxyphenylpyruvate, which is transaminated using glutamate
5-semialdehyde dehydrogenase (which requires NADH or as the nitrogen source to give tyrosine and α-ketoglutarate.
NADPH)  Mammals synthesize tyrosine from the essential amino acid
 Subsequent reduction forms glutamate y semialdehyde phenylalanine (phe), which is derived from food.
 Following spontaneous cyclization is reduced to L- proline  PHENYLALANINE HYDROXYLASE converts phenylalanine to
tyrosine
 If the diet contains adequate amount of nutritionally essential
AA phenylamine- tyrosine becomes non-essential
 However since that reaction is IRREVERSIBLE- dietary tyrosine
cannot replace phenylalanine
 Phenylketonuria (PKU)- deficiency of PHENYLALANINE
HYDROXYLASE causing hyperphenylalanemia and deficiency
of tyrosine
 Phenylalanine Hydroxylase
 Incorporates one atom of O2 into phenylaline and reduces
the other atom to water
 Reducing power is provided as tetrahydrobiopterin, which
derives from NADPH

Figure 14. Conversion of Proline from Glutamate

H. Cysteine

 while not nutritionally essential, it is formed from methionine

which is nutritionally essential
 Biosynthesis begins with the amino acid serine. The sulfur is
derived from methionine, which is converted to homocysteine
through the intermediate S-adenosylmethionine
 Methionine is converted to homocysteine and conjugate to
serine forming cystathionine whose hydrolysis forms cysteine
and homoserine
 STEPS:
 Methionine is converted to to homocysteine

B-synthase)

lyase)
 The sulfur of cysteine is derived from methionine and the
carbon skeleton of serine Figure 16. Conversion of Phenylalanine to Tyrosine

BIOCHEMISTRY 6 of
17
TiTa
Amino Acid Metabolism 1
K. Valine, Leucine, and Isoleucine
J. Hydroxyproline and Hydroxylysine
 ALL are branched-chain, essential AA
 Non-proteinogenic AAs  Tissue Aminotransferases reversibly interconvert all 3 amino
 Occur principally in collagen acids and their corresponding amino acids in the diet
 Since there is no tRNA for either hydroxylated AA, neither  Pyruvate is also a precursor in the synthesis of the branched
dietary hydroxyproline and dietary hydroxylysine is incorporated chain amino acids valine leucine and isoleucine
during protein synthesis
 Peptidyl hydroxyproline and hydroxylysine arise from proline
and lysine, but only after these amino acids have been
incorporated into peptides.
 Hydroxylation of these amino acids is catalyzed by PROLYL
HYDROXYLASE and LYSYL HYDROXYLASE of skin, skeletal
muscle and granulating wounds
 Requires molecular oxygen, ascorbate, Fe ++ and
alphaketogluterate

Figure 19. Branched-Chain AAs Biosynthesis

L. Non-standard AAs
Figure 17. Hydroxyproline from Proline
Selenocysteine
 THE 21st amino acid
 Almost present in all housekeeping enzymes
 Present in some REDOX reactions
 If it is replaced by cysteine, the catalytic activities of the redox
enzymes decrease.
 Example is thioredoxin reductase, glutathione peroxidase and
diodinase
 Implicated in tumoregenesis and atherosclerosis
 Biosynthesis requires cysteine, selenite, ATP a specific tRNA
and several enzymes
 Serine provides carbon skeleton of selenocysteine
 An essential active site residue in mammalian enzymes arises
by cotranslational insertion from modified tRNA

Pyrrolysine
 The 22nd Amino Acid
 Important component of methyltransferases
 This amino acid is encoded by UAG (normally a stop codon),
and its synthesis and incorporation into protein is mediated via
the biological machinery encoded by the pylTSBCD cluster of
genes.
 Pyrrolysine is synthesized in vivo by joining two molecules of L-
lysine. One molecule of lysine is first converted to (3R)-3-methyl-
Figure 18. Hydroxyproline and Hydroxylysine Biosynthesis D-ornithine, which is then ligated to a second lysine. An NH2
group is eliminated, followed by cyclization and dehydration step
to yield L-pyrrolysine+
NOTES:
A deficiency of Vit. C required for the hydroxylation of these NOTES:
amino acids results in Scury which results in bleeding Housekeeping enzymes are ubiquitously present in almost
gums, swelling joints, impaired wound healing results from all living beings to perform essential metabolic functions for
impaired collagen stability. the purpose of survival.

BIOCHEMISTRY 7 of
17
TiTa
Amino Acid Metabolism 1
IV. Summary: Amino Acids Derived from  Zero Nitrogen Balance
Intermediates  In normal adults, nitrogen intake equals nitrogen excretion so
that there is no ammia left that can be toxic to the body
 Derived from Intermediates of Glycolysis  Positive Nitrogen Balance
 Serine  Excess of ingested over excreted nitrogen (occurs when the
 Glycine body uses high amounts of protein)
 Cysteine  Occurs when recovering from trauma
 Alanine  Accompanies growth and pregnancy (higher utilization of
 Derived from Intermediates of TCA Cycle protein
 From a-ketoglutarate  Rapid growth during infancy and adolescent stages
 Glutamate  Pregnancy because there is another individual growing
 Proline inside the mother’s body
 Histidine  Negative Nitrogen Balance
 Glutamine  Nitrogen output exceeds intake
 Arginine  Seen in surgery advanced cancer, kwashiorkor, marasmus,
 From oxaloacetate fasting, starvation and poorly controlled diabetes mellitus
 Aspartate (undersupply of protein)
 Arginine

NOTES:
 Ammonia derived from α-amino nitrogen of amino acids is
highly toxic
 however, ammonia is converted to the amide nitrogen of
the non-toxic amino acid glutamine in tissues
 subsequent deamination of glutamine in the liver releases
ammonia, which is efficiently converted to the non-toxic urea
 compromised liver, cirrhosis and hepatitis, leads to
elevated blood ammonia levels (hyperammonemia)
 Clinically if the liver is compromised, it will lead to
manifestations due to elevated ammonia level

B. Protein Turnover

 Continuous degradation and synthesis of cellular proteins occur

in all forms of life
 Humans turn over 1-2% of total body proteins (principally
muscle protein) daily
Figure 20. Derivations of AAs  High rates of protein degradation occur in tissues that are
undergoing structural rearrangement
V. Protein Catabolism  E.g.: uterine tissue during pregnancy, skeletal muscle in
starvation, and tadpole tail tissue during metamorphosis.
A. Biomedical Importance  Approximately 75% of AA liberated by protein degradation are
reutilized, while the remaining excess free AA are not stored for
future use.
 Amino acids not immediately incorporated into new protein are
rapidly degraded.
 Majority of carbon skeletons of AA are converted to amphibolic
intermediates while the nitrogen in AA is converted to urea and
excreted in urine.

Figure 21. Summary of Nitrogen Balance Figure 23. Protein Turnover

BIOCHEMISTRY 8 of
17
TiTa
Amino Acid Metabolism 1
C. Proteases and Peptidases
 Half-life (t1/2): relative susceptibility of a protein to degradation
 The time required to lower its concentration to half the initial
value
 Average half-life of liver proteins = 30 minutes up to 150
hours+
 Housekeeping enzymes (slow half-life) such as enzymes of
glycolysis: t1/2 values over 100h
 Key regulatory protein (fast half-life) have t1/2 values as low
as 0.5 to 2 hrs
 PEST sequences
 Regions rich in proline (P), glutamate (E), serine (S), and
threonine (T)
 They target some proteins for rapid degradation.
 Intracellular Proteases
 Endopeptidases
 Hydrolyze internal peptide bonds then degrade the
resulting peptides into AA
 Aminopeptidases and carboxypeptidases
 Remove AA sequentially from amino- and carboxy-
termini, respectively

D. Degradation of Proteins Figure 24. Protein Degradation

ATP-Independent Degradation Notes (2020B):

 Blood glycoproteins Avner Ciechanover and Avram Hershko of Israel & Irwin
 degraded after the loss of a SIALIC ACID MOIETY from the Rose of US
non-reducing ends of its oligosaccharide chain -discovered ubiquitin-mediated protein degradation
 Asialoglycoproteins -won Chemistry Nobel Prize 2004
 internalized by liver cell asialoglycoprotein receptors and Metabolic diseases
degraded by lysosomal proteases Extracellular, membrane- -caused by defects in the genes that encode ubiquitin,
associated, and longlived intracellular proteins ubiquitin ligases, or deubiquitinating enzymes
 degraded in lysosomes by ATP-independent process -Angelman Syndrome: autosomal recessive Parkinson’s
disease
ATP- and Ubiquitin-Dependent Degradation -Autosomal recessive Parkinson’s disease
 These are regulatory proteins with short half-lives and of -Von hippel Lindau Syndrome: defect in ubiquitin E3 ligase
abnormal or misfolded proteins. and congenital polycythemia
 Degradation of regulatory proteins with SHORT HALF LIFE and
of abnormal or MISFOLDED proteins occurs in the CYTOSOL
and requires ATP and ubiquitin E. Maintenance of Amino Acid Levels
 Three enzyme system that adds ubiquitin to proteins for  Maintenance of steady-state concentrations of circulating
degradation: plasma AA between meals depends on the net balance from
 E1 (several types) activating enzyme endogenous CHON stores and utilization by various tissues.
 E2 (500 types) a ligase  Muscles: generates half of the total body pool of the free
 E3 (catalyzes the transfer of Ub) a transferase amino Acids
 UBIQUITIN  Liver: site of the urea cycle enzymes necessary for disposal
 Present in all eukaryotic cells of excess nitrogen
 Small 8.5 kDa, 76 residues that target or tag many  Intestine: low levels of activity of these enzymes
intracellular proteins and degradation  Muscle and liver: play major roles in maintaining circulating
 Primary structure is highly conserved amino acid levels
 It is attached by non α peptide bonds formed between the  Free AA especially Alanine and Glutamine (major carriers of
carboxyl terminal of U and the ε amino groups of lysyl Nitrogen in the blood) are released from the muscle to
residues in the target proteins circulation
 The residue present at its amino terminal affects whether or not
a protein is UBIQUITINATED or not
F. Alanine
 Amino terminal Met and Ser retards the reaction while
terminal Asp and Arg accelerates the reaction  KEY gluconeogenic AA
 Soluble proteins undergo POLYUBIQUITINATION  Vehicle of nitrogen transport in the plasma that is extracted
 a ligase catalyzed attachment of four or more additional primarily by the liver
ubiquitin molecules  The rate of hepatic gluconeogenesis from alanine is far
 Subsequent degradation of ubiquitin-tagged proteins usually higher than from all AAs
take place in the PROTEASOME  The capacity of the liver for Gluconeogenesis from alanine
does not reach saturation until the alanine concentration
reaches its 20-30x normal physiologic levels.

BIOCHEMISTRY 9 of
17
TiTa
Amino Acid Metabolism 1
After a meal
 Splanchnic tissues release amino acids
 Peripheral muscles extract amino acids
 Branched chain AA serve special role in N metabolism: fasting
the brain, they serve as energy source and after feeding, they
are extracted by the muscle having spared by the liver.

Figure 25. Glucose-Alanine Cycle

G. Glutamine

Table 2. Type of Organic Waste per Organism

Figure 27. Interorgan relationship of AA metabolism during Fed State
Ammonotelic – in AQUEOUS environment; permits them to
(fishes) excrete water continuously to facilitate Animals Convert α-Amino Nitrogen to Varied End
excretion of Ammonia (highly toxic) Products
 Different animals excrete excess nitrogen as ammonia, as uric
Uricotelic conserve both water and maintain low weight.
acid, or as urea. o Depends on their ecological niche and
(birds) Excrete nitrogen rich uric acid as semisolid
physiology
GUANO
ESTIONS
Ureotelic (land excrete non-toxic highly water-soluble urea VI. UREA CYCLE
animals, High blood urea levels in renal disease are a A. Urea Biosynthesis
humans) consequence, not a cause of impaired renal
function.  Urea
 Major nitrogenous excretory product
 Exits the body in the urine
 Pivotal role in metabolism of amino acids  Ammonia
 Extracted by the gut and the kidney, both of which convert to  Toxic to the brain and CNS
alanine  Removed from the body in the form of urea
 Serve as source of ammonia for excretion to the kidney  Conversion of ammonia to urea occurs in the liver
 Kidney: source of serine for uptake of peripheral tissues  4 Stages in Urea Biosynthesis:
including liver and muscle Branched-chain amino acid (Valine)  Transamination
released by muscle and taken up by brain  Oxidative Deamination of Glutamate
 Ammonia Transport
 Reactions of the urea cycle
H. Overview of Amino Acid Catabolism

Figure 26. Interorgan relationship of AA metabolism Figure 28. Overview of Urea Synthesis

BIOCHEMISTRY 10 of
17
TiTa
Amino Acid Metabolism 1
B. Four Stages of Urea Biosynthesis II. Ammonia Intoxication
 Life Threatening
 Any ammonia produced by the liver is converted to UREA
Transamination  NH3 (Ammonia) can cross cell membrane
 Major process of removing nitrogen from AA  Hepatic Bypass can lead to toxicity
 The nitrogen is transferred as an amino group from the  Clinical Manifestation: Tremor, Slurred speech, blurred vision,
original AA and corresponding alpha ketoglutarate coma -> death
 Interconversion of pairs of α-amino acids and α-keto acids  It reacts with a-ketoglutarate to form glutamate which impairs
 Freely reversible TCA cycle because of depletion of a-ketoglutarate
 Also functions in amino acid biosynthesis
 Pyridoxal phosphate is a required co-factor which is derived Oxidative Deamination of Glutamate
from Vitamin B6
 All amino acids participate in transamination, EXCEPT:
 Lysine, Threonine, Proline, Hydroxyproline
 Involved in both synthesis and degradation

I. Ping-pong Mechanism for Transamination

 Transamination occurs via a “ping-pong” mechanism
characterized by the alternate addition of a substrate and
release of a product. Following removal of it’s a-amino nitrogen
by transamination, the remaining carbon skeleton of an amino
Figure 30. Transdeamination
acid is degraded by pathways
 Pyridoxal phosphates (PLP)
 Often called as Transdeamination
 Derivative of Vitamin B6
 Conversion of α-amino nitrogen to ammonia by the concerted
 Present in all catalytic site of all aminotransferases
action of glutamate aminotransferase and GDH
 Plays a key role in catalysis
 Transfer of amino nitrogen to α-ketoglutarate forms L-glutamate
 Serves as a carrier of amino group during transamination
 Hepatic L-glutamate dehydrogenase (GDH), which can use
 Enzyme-bound shiff base formed between the oxo group of either NAD+ or NADP+, releases this nitrogen as ammonia.
enzyme bound PLP and the alpha amino group of an alpha
 Liver GDH activity is allosterically inhibited by ATP, GTP, and
amino acid
NADH, and is activated by ADP.
 Can rearrange
 The GDH reaction is freely reversible, and also functions in
 In transamination, rearrangement forms an a-keto acid and amino acid biosynthesis
enzyme bound pyridoxamine phosphate
 Transfer of amino acid nitrogen to a-ketoglutarate forms L-
Notes:
glutamate
Glutamate is oxidatively deaminated to α-ketoglutarate &
 Hepatic I-Dehydrogenase (GDH) reaction which can use NAD+
NH3
or NADP+ releases nitrogen as ammonia
 Action is called TRANSDEAMINATION
 Freely Reversible Ammonia Transport
 Ammonia is transported from the peripheral tissue to liver (for its
ultimate conversion to urea) as glutamine or alanine.

I. Glutamine Synthetase
 Formation of glutamine is catalyzed by mitochondrial glutamine
synthase
 Combine ammonia with glutamate to form glutamine, a nontoxic
transport form of ammonia
 Provides glutamine to serve as a carrier of nitrogen, carbon and
energy between organs
 Also play a major role in ammonia detoxification and acid-base
homeostasis
 The glutamine is transported in the blood to the liver where it is
cleaved by glutaminase to produce glutamate and free
ammonia

II. Glutaminase
 Catalyze the hydrolytic release of the amide nitrogen of
glutamine as ammonia.
 Strongly favors glutamate formation
 There are two human isoforms of mitochondrial glutaminase,
termed liver-type and renal-type glutaminase.
 Hepatic glutaminase levels rise in response to high protein
intake while renal kidney type glutaminase increases in
metabolic acidosis. Hydrolytic relaease of the amide nitrogen of
glutamine as ammonia, catalysed by glutaminase, strongly
favors glutamate formation. An analogous reaction is catalysed
Figure 29. Transamination by L-asparigase.

BIOCHEMISTRY 11 of
17
TiTa
Amino Acid Metabolism 1
 The concerted action of glutamine synthase and glutaminase Urea Cycle
thus catalyzes the interconversion of free ammonium ion and
glutamine  “Krebs-Henseleit Cycle”
 Cyclic process
 Synthesis of 1 mole of urea requires
 3 moles of ATP
 1 mole of ammonium ion
 1 mole of aspartate
 5 enzymes, and N-acetyleglutamate (NAG) as an enzyme
activator
 The major metabolic role of ornithine, citrulline, and
argininosuccinate in mammals is urea synthesis. While
ammonium ion, CO2, ATP, and aspartate are consumed, the
ornithine consumed in reaction 2 is regenerated in reaction 5.
Thus, there is no net loss or gain of ornithine, citrulline,
argininosuccinate, or arginine.
 The expression in liver of the RNAs for all the enzymes of the
urea cycle increases several folds in starvation, probably
secondary to enhanced protein degradation to provide energy.
 Some reactions of urea synthesis occur in the matrix of the
mitochondrion, and other reactions in the cytosol

Notes:
Nitrogen enters urea cycle as Ammonium and Aspartate

I. Enzymes involved in Urea Cycle

A. Carbamoyl Phosphate Synthase 1
 Rate-limiting/pacemaker enzyme of the urea cycle
 Found in the mitochondria
 Active only in the presence of N-acetylglutamate, an allosteric
activator that enhances the affinity of the synthase for ATP
 The steady-state level of N-acetylglutamate is dictated by the
balance between its rate of synthesis from acetyl-CoA and
glutamate and its rate of hydrolysis to acetate and glutamate,
reactions catalyzed by N-acetylglutamate synthase (NAGS)
and Nacetylglutamate deacylase (hydrolase), respectively.
 Acetyl-CoA + l-glutamate → N-acetyl-l-glutamate +
CoASH
 N-acetyl-l-glutamate + H2O → l-glutamate + acetate
 Catalyze the condensation of CO2, ammonia, and ATP to form
carbamoyl phosphate
 ammonia provides the 1st nitrogen of urea
 synthesis of 1 mol of this enzyme requires 2 ATPs
 The reaction proceeds stepwise.
 this step of the cycle occurs within the inner mitochondria

Table 3. CPS I vs CPS II

Notes:
A cytosolic form of this enzyme, carbamoyl phosphate
synthase II, uses glutamine rather than ammonia as the
Figure 31. Ammonia Metabolism
nitrogen donor and functions in pyrimidine biosynthesis
BIOCHEMISTRY 12 of
17
TiTa
Amino Acid Metabolism 1
D. Orthinine Transcarbomylase  Definitive diagnosis involves quantitative assay of the activity of
 Catalyzes transfer of the carbamoyl group of carbamoyl the enzyme suspected to be defective.
phosphate to ornithine, forming citrulline and orthophosphate  The DNA sequence of the gene that encodes a given mutant
 While the reaction occurs in the mitochondrial matrix, both the enzyme is compared to that of the wild-type gene
formation of ornithine and the subsequent metabolism of  To identify the specific mutation(s) that cause the disease.
citrulline take place in the cytosol.  The exponential increase in DNA sequencing of human genes
 Membrane permeases then allow citrulline to be translocated has identified dozens of mutations of an affected gene that are
from the inner mitochondrial membrane to the cytosol benign or are associated with symptoms of varying severity of a
given metabolic
Notes:
Entry of ornithine into mitochondria and exodus of citrulline
from mitochondria therefore involve mitochondrial inner Notes:
membrane permeases Most common symptoms include vomiting, failure to feed,
muscle weakness, lethargy, sleepiness, due to accumulation
C. Arginosuccinate Synthase of ammonia
 links aspartate and citrulline, forming arginosuccinate “usually symptoms include lethargy or sleepiness
 provides the 2nd nitrogen of urea (somnolence) because ammonia is toxic to the brain”
 requires the use of 1 mol ATP and forms an intermediate,
citrullyl-AMP
 Subsequent displacement of AMP by aspartate then forms A. Carbamoyl Phosphate Synthetase 1
argininosuccinate.
 Defects lead to Hyperammonemia type 1
D. Arginosuccinate lyase  Typical symptoms include very high ammonium levels in the
 Catalyze the cleaves arginosuccinate, forming arginine and blood and mental retardation
fumarate  NAG is essential for the activity of CPS 1
 Retain all three nitrogens in arginine and release the aspartate  Rare; 1 : 62,000
skeleton as fumarate
 Fumarate links the urea cycle to the TCA B. N-acetylglutamate Synthase
 addition of H2O to fumarate forms L-malate, which in turn forms  D
oxaloacetate after NAD-dependent oxidation  Therefore, deficiency also exhibits same symptoms as CPS 1
 These two reactions are analogous to reactions of the citric defects
acid cycle, but are catalyzed by cytosolic fumarase and  Responds to administration of N-actyleglutamate
malate dehydrogenase
 Transamination of oxaloacetate by glutamate aminotransferase
C. Ornithine Permease
then re-forms aspartate.
 The carbon skeleton of aspartate-fumarate thus acts as a  Deficiency results in HHH syndrome (Hyperornithemia,
carrier of the nitrogen of glutamate into a precursor of urea. Hyperammonemia, Homocitrullinuria)
 Results from the mutation of the ORNT1 gene
E. Arginase  Failure of urea cycle because of inability to translocate cytosolic
 catalyzes the hydrolytic cleavage of the guanidino group of ornithine back into mitochondria
arginine to release urea  In the absence of its normal acceptor (ornithine), mitochondrial
 this cleavage results in the release of urea as well as the carbamoyl phosphate carbamoylates lysine to homocitrulline,
formation of ornithine resulting in homocitrullinuria.
 ornithine re-enters liver mitochondria and begins the cycle again
 this same ornithine molecule is regenerated from the 2 nd D. Ornithine Transcarbamoylase
reaction
 X-linked deficiency
 only the NH4+, CO2, ATP, and aspartate are consumed in the
 Results in Hyperammonemia type 2
cycle
 Mothers with this disorder exhibit hyperammonemia and
 Ornithine and lysine are potent inhibitors of arginase, and
aversion to protein-rich food
compete with arginine.
 High levels of glutamine in blood, CSF, and urine due to
 Arginine also serves as the precursor of the potent muscle
enhanced glutamine synthesis in the presence of high levels of
relaxant nitric oxide (NO) in a Ca2+-dependent reaction
ammonia in the tissues
catalyzed by NO synthase

VII. METABOLIC DISORDERS OF THE UREA CYCLE E. Arginosuccinate Synthase

 25x higher Km for citrulline
 Similar or identical clinical signs and symptoms can accompany  fluid citrulline levels are elevated
various genetic mutations in a gene that encodes a given  Blood citrulline: above 1000 uM
enzyme or in enzymes that catalyze successive reactions in a  1-2g of citrulline excreted daily
metabolic pathway.
 Rational therapy is based on an understanding of the relevant
biochemical enzyme-catalyzed reactions in both normal and F. Arginosuccinate Lyase
impaired individuals.  Defects result in argininosuccinicaciduria
 The identification of intermediates and of ancillary products that  Elevated arginosuccinate in blood, CSF, and urine, with friable
accumulate prior to a metabolic block tufted hair or trichorrhexis nodosa
 Basis for metabolic screening tests to implicate the reaction  Blood citrulline: 100-300 uM
that is impaired.  Diagnosed via measurement of erythrocyte arginosuccinate
lyase activity on umbilical cord blood or amniotic fluids
BIOCHEMISTRY 13 of
17
TiTa
Amino Acid Metabolism 1

G. Arginase
 Defects result in Hyperargininemia
 An autosomal recessive defect in the gene for arginase
 Symptoms appear at 2-4 years old
 Arginine is elevated in blood and CSF

Figure 32. Urea cycle Defects

VIII. REGULATION OF THE UREA CYCLE

 Urea cycle regulation is dependent on dietary factors and

hormone concentrations
 Ammonia can come from diet, from deamination, or Figure 33. Urea cycle Schematic
bacteria in the GI tract inducing formation of carbamoyl
phosphate by mitochondrial carbamoyl phosphate
synthetase I. Notes:
•↑ dietary protein → ↑ catabolism of amino acids in the
Regulated by substrate availability presence of required enzymes for urea cycle → ↑ urea
 higher rate of ammonia production = higher rate of urea
production of toxic NH3 (hyperammonemia)
 Urea cycle is a disposal pathway which removes the toxic •Prolonged starvation → breakdown of muscle protein → ↑
compound in our bodies urea
 This can also mean that higher protein can also act as a  N-Acetylglutamate (NAG) is an essential activator for CPS I,
feed-forward regulation since it increases urea enzyme levels the rate-limiting step in the urea cycle. It increases the
affinity of CPS I for ATP. NAG is synthesized from acetyl
Feed-forward regulation CoA and glutamate by N-acetylglutamate synthase in a
reaction for which arginine is an activator. The cycle is also
 control of a metabolic pathway by a metabolite of the pathway
regulated by substrate availability (short-term regulation)
that acts in the same direction as the metabolic flux, i.e.
downstream or 'later' in the pathway, e.g. the activation of
pyruvate kinase by fructose 1,6-bisphosphate)
 In contrast to Feedback regulation which produces functional
products IX. CASE

Other Regulators 5 newborns were noted to develop lethargy, hypothermia and

apnea after 24 hours. They all appeared normal at birth. They
 First, synthesis of n-acetyl glutamate, which is the allosteric
developed seizures and crania CT scan revealed brain swelling.
activator of the CPS I, this activator is made in the liver and
Upon examination the serum ammonia is very high with the
intestine when stimulated by available arginine
following lab results:
 Second, arginase is inhibited by ornithine and lysine making it
able to become rate limiting in some cases

BIOCHEMISTRY 14 of
17
TiTa
Amino Acid Metabolism 1
Urine Blood Blood Blood
Infant
Orotate Citrulline Arginine Ammonia Carbamoylphosphate Carbamoylphosphate
synthetase 1 synthetase 2
1 MM Low Low Low High
Cellular Mitochondria Cytosol
High Location
2 SM - Low High
(>1500UM) Pathway Urea cycle Pyrimidine synthesis
Involved
3 FM - - High High
Source of Ammonia Amide group of
4 LB High Low Low High Nitrogen glutamine
High Regulation Activator; N-acetyl- Activator: PRPP
5 CD - Low High
(>200UM) glutamate Inhibitor: UTP

1. Given the following information which urea cycle enzyme will 3. Explain the clinical manifestations of the patient
yeld the result of infant 1,2,3,4, and 5?
Seizures and Brain Swelling

Figure 34. Summary of Urea Cycle Defects

Infant 1: Carbamoyl phosphate synthetase

Infant 2: Arginosuccinate
Infant 3: Arginase
Infant 4: Ornithine Transcarbomylase
Infant 5: Argininosuccinate Lyase

2. Draw the Urea Cycle and the involved enzyme and co-factors

Figure 35. Urea Cycle Figure 36. Effect of high Ammonia

BIOCHEMISTRY 15 of
17
TiTa
Amino Acid Metabolism 1
4. What are the treatment regimens for these patients? 3. Which of the following statements about the urea cycle is
 Protein Restricted Diet and Essential Amino Acid correct?
Supplementation
 Nitrogen Scavengers (Benzoic Acid and Phenylbutyrate) a) Argininosuccinate is lysed to urea and ornithine in the
 Massive Arginine Supplementation urea cycle.
 Dialysis b) Carbamoyl phosphate supplies both of the nitrogen atoms
 Excellent Candidate for Gene Therapy of urea in the urea cycle.
c) The formation of urea from the urea cycle yields energy.
1. Which of the following statements about the metabolism of d) Arginine is hydrolysed to urea and ornithine in the urea
amino acids is correct? cycle.

a) Essential amino acids can be formed from other amino 4. Which metabolic abnormality gives rise to the serious disease
acids supplied in the diet. phenylketonuria?
b) Excess dietary amino acids cannot be converted to other
metabolites. a) Homocysteine cannot be converted into methionine
c) Excess dietary amino acids that cannot be oxidised are b) Phenylalanine cannot be converted into tyrosine
stored as muscle proteins. c) Phenylalanine cannot be converted into alanine
d) Essential amino acids cannot be formed from other d) Tyrosine cannot be converted into phenylalanine
amino acids but must be supplied in the diet. Answers: d, b, d, b

2. Which of the following statements about the role of glutamate XI. REREFENCES
dehydrogenase is correct?
Dr Alba’s powerpoint
a) Glutamate dehydrogenase has an FAD prosthetic group. Harper’s
Baynes
b) Glutamate dehydrogenase oxidatively deaminates
2020 Transes
glutamate producing -ketoglutarate.
Caballar’s powerpoint
Online Journals and Articles

XII. APPENDIX

BIOCHEMISTRY 16 of
17