Biochemistry _ amino acid oxidation

1. CHAPTER 19
Amino Acid Oxidation
Production of Urea
–How proteins are digested in animals
–How amino acids are degraded in animals
–How urea is made and excreted
Key topics:

2. Oxidation of Amino Acids is a Significant
Energy-Yielding Pathway in Carnivores
•Not all organisms use amino acids as the source
of energy
•About 90% of energy needs of carnivores can be
met by amino acids immediately after a meal
•Only a small fraction of energy needs of
herbivores are met by amino acids
•Microorganisms scavenge amino acids from their
environment for fuel

3. Sources and Uses of Amino Acids
Sources
1.Proteins in the diet provide both essential and non-
essential amino acids in contrast to microorganisms that
for the most part synthesize their own.
2.Turnover of endogenous proteins
3.de novo biosynthesis (non-essential amino acids)
Uses
1.Protein synthesis
2.Nitrogen and carbon source of general and special
product biosynthesis
3.Energy source
a.glucogenic (those that can be used for the synthesis of glucose)
b.ketogenic (those whose metabolism leads to ketone bodies)

4. Metabolic Circumstances of
Amino Acid Oxidation
Amino acids undergo oxidative catabolism
under three circumstances:
–Protein amino-acid residues from normal turnover
are recycled to generate energy and molecular
components
–Dietary amino acids that exceed body’s protein
synthesis needs are degraded
–Proteins in the body are broken down to supply
amino acids for catabolism when carbohydrates
are in short supply (starvation, diabetes mellitus),

5. Protein Turnover and Nitrogen
Balance
Protein Degradation:
• Endogenous proteins degrade continuously
- Damaged
- Mis-folded
- Un-needed
• Dietary protein intake - mostly degraded
Nitrogen Balance - expresses the patient’s current
status - are they gaining or losing net Nitrogen?

6. Dietary Proteins are
Enzymatically Hydrolyzed
•Pepsin cuts protein into peptides in the stomach
•Trypsin and chymotrypsin cut proteins and larger
peptides into smaller peptides in the small
intestine
•Aminopeptidase and carboxypeptidases A and B
degrade peptides into amino acids in the small
intestine

7. stomach pancreas to
small intestine
intestinal
wall
pepsin Trypsin
Chymotrypsin
carboxypeptidase A
carboxypeptidase B
elastase
dipeptidases

8. •(a) gastrin -> secretion of
HCl by parietal cells and
pepsin by chief cells
•(b) exocrine cells synthesize
zymogens
–zymogen granules fuse with
plasma membrane
–zymogens released into the
lumen of the collecting duct
–collecting ducts -> pancreatic
duct -> small intestine.
•(c) Amino acids -> villi ->
capillaries
Enzymatic
Degradation of
Dietary Proteins

9. Overview
of Amino
Acid
Catabolism

10. OVERVIEW OF AMINO ACID METABOLISM
ENVIRONMENT ORGANISM
Ingested
protein
Bio-
synthesis
Protein
AMINO
ACIDS
Nitrogen
Carbon
skeletons
Urea

Degradation
(required)
1
2 3
a
b
Purines
Pyrimidines
Porphyrins
c c
Used for
energy pyruvate
α-ketoglutarate
succinyl-CoA
fumarate
oxaloacetate
acetoacetate
acetyl CoA
(glucogenic)(ketogenic)

11. Degradation of amino acids to one of
seven common metabolic intermediates.

12. Amino acid metabolism
•Metabolism of amino acids differs, but 3
common reactions:
–Transamination
–Deamination
–Formation of urea

13. Typical first transamination reaction:
The usual AA acceptor is α-ketoglutarate, producing
GLUTAMATE and the new a-keto acid.
Transamination is a reaction between an amino acid and
a keto-acid in which the amino group is transferred from
the donor amino acid onto the acceptor keto-acid.

14. TRANSAMINATION

15. Enzymatic Transamination
•Typically, a-ketoglutarate
accepts amino groups
•L-Glutamine acts as a
temporary storage of nitrogen
•L-Glutamine can donate the
amino group when needed for
amino acid biosynthesis
•All aminotransferases rely on
the pyridoxal phosphate (PLP)
cofactor

16. Structure of Pyridoxal Phosphate
and Pyridoxamine Phosphate
•Intermediate, enzyme-
bound carrier of
amino groups
•Aldehyde form can
react reversibly with
amino groups
•Aminated form can
react reversibly with
carbonyl groups

17. A small number of amino acids undergo
oxidative or non-oxidative deamination

18. 18
Urea Formation
•Occurs primarily in liver; excreted by kidney
•Principal method for removing ammonia
•Hyperammonemia:
•Defects in urea cycle enzymes
•Severe neurological defects in neonates

19. Treatment of deficiency of Urea Cycle enzymes (depends
on which enzyme is deficient):
limiting protein intake to the amount barely adequate
to supply amino acids for growth, while adding to the
diet the a-keto acid analogs of essential amino acids.
Liver transplantation has also been used, since liver
is the organ that carries out Urea Cycle.
Dialysis
Increase ammonia excretion: Na benzoate, Na
phenylbutyrate, L-arginine, L-citrulline

20. Postulated mechanisms for toxicity of high
[ammonia]:
1. High [NH
3
] would drive Glutamine Synthase:
glutamate + ATP + NH
3
 glutamine + ADP + P
i

This would deplete glutamate – a neurotransmitter &
precursor for synthesis of the neurotransmitter GABA.
2. Depletion of glutamate & high ammonia level would drive
Glutamate Dehydrogenase reaction to reverse:
glutamate + NAD(P)
+
a-ketoglutarate +
NAD(P)H + NH
4
+
The resulting depletion of a-ketoglutarate, an essential
Krebs Cycle intermediate, could impair energy metabolism
in the brain.

21. 21
GABA Formation
NH
3
+
-
O
2CCH
2CH
2CHCO
2
-
NH
3
+
-
O
2CCH
2CH
2CH
2
Glutamate
Gamma-aminobutyrate
(GABA)
GABA is an important inhibitory neurotransmitter in the brain
Drugs (e.g., benzodiazepines) that enhance the effects
of GABA are useful in treating epilepsy
Glutamate
decarboxylase
CO
2

22. •Glutamate is the precursor of free GABA in GABAergic terminals and
comes from two different sources (Kreb's cycle in glia cells and
glutamine in nerve terminals). Next, the enzyme glutamic acid
decarboxylase (GAD) forms GABA from glutamate. After being
released into the synapses, GABA is inactivated by reuptake mediated
by GABA transporters (GATs) into presynaptic terminals or into glia
cells where it is metabolized by GABA-transaminase (GABA-T).

23. N balance = NN balance = N
inin - N - N
outout
1Major dietary source of N is Protein (>95%), since the
diet has very few free amino acids (AA)
2AA are used for Protein Synthesis & N containing
compounds
3 AA in excess are degraded (used for energy)
N is disposed of in urea (80%), ammonia, uric acid or
creatinine in urine with small amounts in fecal matter
(undigested)

24. Nitrogen Acquisition
•Nitrogen Fixation
•Nitrate Assimilation
•Ammonium Assimilation

25. N
2
is converted to metabolically
useful forms (is "fixed") only by a
few species of prokaryotes, called
Diazotrophs.
Diazotrophs of the genus
Rhizobium live symbiotically in the
root nodules of legumes, where
they convert N
2
to NH
3
(ammonia)
in a process called
NITROGEN FIXATION:
NITROGENASE
N
2
+ 8 H+ + 8 e- + 16 ATP + 16 H
2
O → 2 NH
3
+ H
2
+ 16 ADP + 16 Pi

26. * But, less than 1% of N entering the biosphere comes from N
fixation.
Another oxidized form of nitrogen, NO3
-
(nitrate ion) is also
found in the soils and oceans.
It is converted to NH4
+
through NITRATE ASSIMILATION:
* The reduction of NO3
-
to NH4
+
(ammonium ion) occurs in
green plants, various fungi, and certain bacteria in a two-step
pathway:
(1) The 2-electron reduction of nitrate to nitrite:
NO
3
-
+ 2 H
+
+ 2 e
-
→ NO
2
-
+ H
2
O ( catalyzed by nitrate reductase)
(2) This is followed by the 6-electron reduction of nitrite to ammonium:
NO
2
-
+ 8 H
+
+ 6 e
-
→ NH
4
+
+ 2 H
2
O ( catalyzed by nitrite reductase)
*NH
3
/NH
4
+
can be incorporated into the amino acids glutamate
by glutamate dehydrogenase (and glutamine by glutamine
synthetase.

27. Nitrate Assimilation
(Green plants, some fungi and bacteria)
NO
3
–
+ NADH + H
+
NO
2
–
+ H
2O + NAD
+
Nitrate Reductase
NO
2
–
+ 8H
+
+ 6e
–
NH
4
+
+ 2H
2O
Nitrite Reductase

28. Ammonium Assimilation
(Carbamoyl Phosphate Synthetase)
H
2
NC
O
OP
2ATP 2ADP + P
i
NH
3 + HCO
3
–
(Biosynthetic Glutamate Dehydrogenase)
and/or
(Glutamine Synthetase)
NH
3
Glutamate
NH
3
Glutamine

29. No animals are capable of either N-fixation or
nitrate assimilation, so animals are totally
dependent on plants and microorganisms for
the synthesis of organic nitrogenous
compounds, such as amino acids and proteins,
to provide this essential nutrient.

30. Nitrogen balance = nitrogen ingested - nitrogen excreted
(primarily as protein) (primarily as urea)
Nitrogen balance = 0 (nitrogen equilibrium)
protein synthesis = protein degradation
Positive nitrogen balance
protein synthesis > protein degradation
Negative nitrogen balance
protein synthesis < protein degradation
Nitrogen balance
•Protein content of adult body remains remarkably constant
–Protein constitutes 10-15% of diet
•Equivalent amount of amino acids must be lost each day

31. Fates of Nitrogen in Organisms
•Plants conserve almost all the nitrogen
•Many aquatic vertebrates release ammonia to their
environment
–Passive diffusion from epithelial cells
–Active transport via gills
•Many terrestrial vertebrates and sharks excrete nitrogen
in the form of urea
–Urea is far less toxic that ammonia
–Urea has very high solubility
•Some animals, such as birds and reptiles excrete
nitrogen as uric acid
–Uric acid is rather insoluble
–Excretion as paste allows to conserve water
•Humans and great apes excrete both urea (from amino
acids) and uric acid (from purines)

32. Excretory
Forms of
Nitrogen

33. The Amino
Group is
Removed From
All Amino Acids
First

34. Glutamate can Donate
Ammonia to Pyruvate to
Make Alanine
•Vigorously working muscles
operate nearly anaerobically
and rely on glycolysis for
energy
•Glycolysis yields pyruvate that
muscles cannot metabolize
aerobically; if not eliminated
lactic acid will build up
•This pyruvate can be
converted to alanine for
transport into liver

35. Ammonia in
Transported in the
Bloodstream Safely
as Glutamate
•Un-needed glutamine
is processed in
intestines, kidneys
and liver

36. Excess Glutamate is Metabolized in
the Mitochondria of Hepatocytes

37. The Glutamate
Dehydrogenase
Reaction
•Two-electron oxidation
of glutamate followed
by hydrolysis
•Net process is
oxidative deamination
of glutamate
•Occurs in mitochondrial
matrix in mammals
•Can use either NAD
+
or
NADP
+
as electron
acceptor

38. Nitrogen
from
Carbamoyl
Phosphate
Enters the
Urea Cycle

39. •Urea is produced in the Urea is produced in the liverliver
•From the liver, it is transported in the blood to the From the liver, it is transported in the blood to the kidneyskidneys for for
excretion in urine excretion in urine
Urea is composed of:Urea is composed of:
Two nitrogen atomsTwo nitrogen atoms
•First nitrogen atom is from free ammoniafree ammonia
•Second nitrogen atom is from aspartateaspartate
Carbon & oxygen atoms are from COCarbon & oxygen atoms are from CO22
Urea Cycle

40. The Reactions in the Urea Cycle
•1 ornithine + carbamoyl phosphate => citrulline
–(entry of the first amino group).
–citrulline passes into the cytosol.
•2a citrulline + ATP => citrullyl-AMP + PPi
•2b citrullyl-AMP + Aspartate => argininosuccinate + AMP
–(entry of the second amino group).
•3 argininosuccinate => arginine + fumarate
–fumarate enters the citric acid cycle.
•4 arginine => urea + ornithine
–Ornithine passes to the mitochondria to continue the cycle

41. Urea
Cycle N-2
from
Aspartate

42. Ammonia is Re-captured via
Synthesis of Carbamoyl Phosphate
•This is the first nitrogen-acquiring reaction

43. Entry of Aspartate into the Urea
Cycle
•This is the second nitrogen-acquiring reaction

44. Aspartate –Argininosuccinate Shunt
Links Urea Cycle and Citric Acid Cycle

45. Hereditary deficiency of any of the Urea Cycle
enzymes leads to hyperammonemia - elevated
[ammonia] in blood.
Total lack of any Urea Cycle enzyme is lethal.
Elevated ammonia is toxic, especially to the
brain.
If not treated immediately after birth, severe
mental retardation results.

46. Fate of UreaFate of Urea
Urea Urea
(synthesized in the liver) (synthesized in the liver)
BloodBlood
KidneyKidney intestine


Urine cleaved by bacterial urease
AmmoniaAmmonia CO2
In stool Reabsorbed in blood

48. 1- Urea Urea
in the liverin the liver
•is quantitatively the most important most important disposal route for
ammonia
•Urea is formed in the liver liver from ammonia (urea cycle)
•UreaUrea travels in the blood from the liver to the kidneyskidneys
where it is filtered to appear in urineurine

Disposal of Ammonia

49. 2- GGlutamine lutamine
in in most peripheral tissues most peripheral tissues especiallyespecially brain, Skeletal Muscles brain, Skeletal Muscles
& liver& liver
•In most peripheral tissues, glutamate binds with ammoniaammonia by action of
glutamine synthase glutamine synthase
•in the brainbrain, it is the major mechanism of removal of ammonia from the
brain
•This structure provides a nontoxic storage & transport form of ammonia nontoxic storage & transport form of ammonia
•Glutamine is transported to blood to other organs esp. liver & kidneys
•In the liver & Kidney, glutamine is converted to ammonia & glutamate
by the enzyme glutaminaseglutaminase.
Disposal of Ammonia

50. 3- Alanine Alanine
in skeletal musclesin skeletal muscles
• AmmoniaAmmonia + Pyruvate form alanine alanine in skeletal muscles
• Alanine is transported in blood to liver
• In liver, alanine is converted to pyruvate & ammoniaammonia
• Pyruvate can be converted to glucoseglucose (by gluconeogenesis)
•GlucoseGlucose can enter the blood to be used by skeletal muscles
(GLUCOSE - ALANINE PATHWAY)(GLUCOSE - ALANINE PATHWAY)

Disposal of Ammonia

51. Alanine Alanine
in Skeletal Musclesin Skeletal Muscles
GlutamineGlutamine
in Most Tissuesin Most Tissues
Esp. brain & KidneysEsp. brain & Kidneys
UreaUrea
in Liverin Liver
Disposal of Ammonia

52. Not All Amino Acids can be
Synthesized in Humans
•These amino acids
must be obtained
as dietary protein
•Consumption of a
variety of foods
(including
vegetarian only
diets) well supplies
all the essential
amino acids

53. Essential amino acids
Mammalian cells lack enzymes to synthesize their carbon
skeletons (a-keto acids).
Isoleucine, leucine, & valine
Lysine
Threonine
Tryptophan
Phenylalanine (Tyr can be made from Phe.)
Methionine (Cys can be made from Met.)
Histidine (Essential for infants.)

54. 54
V Valine
F Phenylalanine
W Tryptophan
I Isoleucine
T Threonine
H Histidine
M Methionine
L Leucine
K Lysine
•One way to remember the 9 essential amino acids is with the
mnemonic VF WITH MLK (Very Full With Milk):

55. Fates of carbon
skeleton of
amino acids

56. Fate of Individual Amino Acids
•Seven to acetyl-CoA
–Leu, Ile, Thr, Lys, Phe, Tyr, Trp
•Six to pyruvate
–Ala, Cys, Gly, Ser, Thr, Trp
•Five to a-ketoglutarate
–Arg, Glu, Gln, His, Pro
•Four to succinyl-CoA
–Ile, Met, Thr, Val
•Two to fumarate
–Phe, Tyr
•Two to oxaloacetate
–Asp, Asn

57. Glucogenic vs ketogenic amino acids
•Glucogenic amino acids
(are degraded to pyruvate or
citric acid cycle
intermediates) - can supply
gluconeogenesis pathway
•Ketogenic amino acids (are
degraded to acetyl CoA or
acetoacetyl CoA) - can
contribute to synthesis of
fatty acids or ketone bodies
•Some amino acids are both
glucogenic and ketogenic

58. Summary of
Amino Acid
Catabolism

59. 6 Amino Acids ->
Pyruvate
Ala, Gly, Ser,
Cys,Trp,Thr.

60. 7 AAs -> Acetyl CoA [W,K,F,Y, L]

61. I, M, T, V->
Succinyl-CoA

62. Albinism –
genetically determined
lack or deficit of enzyme
tyrosinase
Tyrosinase in
melanocytes oxidases
tyrosine to DOPA and
DOPA-chinone
tyrosinase
Phenylalanine
Tyrosine Tyroxine
Melanin
DOPA
Dopamine
Norepinephrine
Epinephrine

63. The pathways for the biosynthesis of amino acids are diverse
Common feature: carbon skeletons come from
intermediates of
 glycolysis,
 pentose phosphate pathway,
 citric acid cycle.
All amino acids
are grouped
into families
according to the
intermediates
that they are
made from

65. Summary
•Amino acids from protein are an important energy source
in carnivorous animals
•Catabolism of amino acids involves transfer of the amino
group via PLP-dependent aminotransferase to a donor
such as a-ketoglutarate to yield L-glutamine
•L-glutamine can be used to synthesize new amino acids,
or it can dispose of excess nitrogen as ammonia
•In most mammals, toxic ammonia is quickly recaptured
into carbamoyl phosphate and passed into the urea cycle

66. Sample question
•The site of amino acid catabolism is the:
A. Stomach
B. Small intestine
C. Large intestine
D. Liver

67. Sample question
•The first step in the catabolism of most amino
acids is
•A. Removal of carboxylate groups
•B. Enzymatic hydrolysis of peptide bonds
•C. Removal of the amino group
•D. Zymogen cleavage

68. Sample question
Which of the following is true of urea?
•A. more toxic to human cells than ammonia
•B. the primary nitrogenous waste products of
humans.
•C. insoluble in water
•D. the primary nitrogenous waste product of
most aquatic invertebrates

69. Sample question
A glucogenic amino acid is one which is
degraded to
•A.keto-sugars
•B.either acetyl CoA or acetoacetyl CoA
•C.pyruvate or citric acid cycle
intermediates
•D.none of the above

70. Sample question
Transamination is the process where
•A.carboxyl group is transferred from amino
acid
•B.α-amino group is removed from the amino
acid
•C.polymerization of amino acid takes place
•D.none of the above

71. Sample question
Transamination is the transfer of an amino
•A.acid to a carboxylic acid plus ammonia
•B.group from an amino acid to a keto acid
•C.acid to a keto acid plus ammonia
•D.group from an amino acid to a carboxylic
acid