Amino Acid Metabolism: Objectives
1. Few questions…
a. Which amino acids are the essential ones and which are nonessential?
i. Essential – there are no biosynthetic pathways or not synthesized in adequate
quantities
1. Arginine **Conditionally essential (can be made from glutamate +
aspartate)
2. Histidine **Conditionally essential
3. Isoleucine
4. Leucine
5. Lysine
6. Methionine
7. Phenylalanine
8. Theronine
9. Tryptophan
10. Valine
ii. Nonessential
1. Alanine (made from pyruvate and an amino acid)
2. Asparagine (made from aspartate + glutamine)
3. Aspartate (made from oxaloacetate)
4. Cysteine **Made from methionine (+ serine)
5. Glutamate (made from aspartate + alpha-ketogluterate)
6. Glutamine (made from glutamate and ammonia)
7. Glycine (made from threonine)
8. Proline (made from glutamate)
9. Serine (made from glutamate or glycine or choline)
10. Tyrosine**Made from phenylalanine
b. Which nonessential amino acids are derived directly from an essential amino acid?
i. They are readily made from intermediates of central pathways
c. Which amino acids are conditionally essential and why?
i. Arginine – not essential if have adequate glutamate and aspartate
ii. Histidine – essential in infants (pathway is present as age)
d. Methionine: need in higher amounts (solely responsible for making Cysteine)
e. Phenylalanine: need in higher amounts (solely responsible for making Tyrosine)
2. How are amino acids absorbed in small intestine?
a. Amino acids are taken up by enterocytes in the small intestine
i. AAs transfer through enterocytes to the portal circulation by diffusion,
facilitated diffusion, or active transport
3. What molecules serve as carriers of nitrogen?
a. Glutamine (in a number of pathways)
b. Alanine (inter-organ transfer of nitrogen…as in glucose-alanine cycle)
c. Urea (transfers N in blood for excretion in the urine is its ONLY function)
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Amino Acid Metabolism: Objectives
4. What properties of urea make it well suited as a carrier of nitrogen?
a. Urea is nontoxic.
b. Urea is highly water soluble.
c. Its only purpose in life is to excrete excess Nitrogen out of the body and into the toilet.
5. Explain how transaminases and glutamate dehydrogenase play a central role in nitrogen
metabolism.
a. N is carried in blood serum as an amino group on amino acids
i. Primarily, alanine and glutamine
b. Transaminases: transfer amino groups to alpha-ketogluterate to make glutamate
i. Amino Acid + alpha-ketogluterate  keto acid + Glutamate
c. Glutamate dehydrogenase: releases ammonia for urea synthesis
i. Glutamate + NADP +H2O  alpha-ketogluterate + NADPH + NH3
1. Enzyme: glutamate dhd
d. Thus, a free NH3 is formed… which can be brought together with HCO3- and attach to
urea to be excreted
i. NH3 + HCO3-  Urea to be excreted
6. Under what conditions are ammonium ions excreted in relatively high concentrations in the
urine?
a. During metabolic acidosis:
i. Ex: Ketoacidosis due to uncontrolled diabetes mellitus is most common
ii. Urea production is suppressed
iii. Ammonium is excreted directly by the kidney!
b. Glutamine + H2O  Glutamate +NH4+
i. What enzyme plays an important role under these conditions?
1. Glutaminase
7. Explain how the glucose alanine cycle operates in inter-organ metabolism. Why is this cycle
important?
a. Provides for the nontoxic transport of nitrogen (produced by metabolizing amino acids)
from skeletal muscle
b. Produces pyruvate for the production of glucose by gluconeogenesis (in the liver)
i. Especially useful in times of fasting or starvation
Glucose Alanine Cycle
Blood
Amino acid
keto acid
Liver
Muscle
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Amino Acid Metabolism: Objectives
8. Explain how pyridoxal phosphate functions in amino group transfer by transaminases.
a. Pyridoxal phosphate acts as an electrophile for covalent catalysis
i. Essential for the transfer of N (as an amine group) that goes eventually to urea
b. Pyridoxal phosphate and transaminase attach
i. Then, transaminase and the amino acid switch places
1. So the pyridoxal phosphate is attached now to the amino acid
ii. Release of the amino acid, and the free pyridoxal phosphate are produced.
c. THUS: Pyridoxal phosphate acts as an electrophile
9. How are the reactions of the urea cycle compartmentalized inside of cells?
a. Occurs in the Kidney and the Liver:
i. Occurs within the mitochondria:
1. HCO3- + NH3 + 2 ATP  Carbamoyl Phosphate
a. Enzyme: Carbamoyl phosphate synthetase I
2. Ornithine + Carbamoyl phosphate  Citrulline and PO4H2a. Ornithine needs a membrane carrier to enter or exit the
mitochondria
i. Ornithine is made in the cytosol
ii. Occurs within the cytosol:
1. Citrulline + Aspartate + ATP  Argininosuccinate
a. Citrulline is transported into the cytosol (via membrane carrier)
2. Argininosuccinate  Fumerate + Arginine
b. In the liver, the reactions will continue:
i. Arginine + H2O  Ornithine + Urea
NH3 + HCO3-
Carbamoyl phosphate
synthetase I
2 ATP
1
2 ADP + Pi
Carbamoyl PO4
PI
Ornithine
transcarbamylase
2
Mitochondrial
Matrix
Urea Cycle
Summary
Aspartate
Ornithine
Citrulline
Ornithine
Citrulline
Urea
Argininase
Aspartate
5
ATP
3
Agininosuccinate
synthetase
AMP + PPi
H2O
Arginine
Cytosol
Glutamate
4
argininosuccinate
Fumarate
Argininosuccinate
lyase
3
Glutamate
Amino Acid Metabolism: Objectives
10. Explain the role of the shuttles for ornithine/citrulline and malate/aspartate in the urea cycle.
a. Glutamate is an anti-transporter with Aspartate
i. Aspartate into the cytosol, glutamate into the mitochondria
b. Alpha-ketogluterate is an anti-transporter with Malate
i. Alpha-ketogluterate into the cytosol, malate into the mitochondria
c. Citrulline and ornithine are antiports as well
i. Citrulline into the cytosol, ornithine into the mitochondria
d. See #9 diagram for more info…
11. How are the urea cycle and the TCA cycle linked?
a. Malate (from Fumerate)
b. Aspartate
c. Alpha-ketogluterate formation (can enter at that point in the TCA)
Links Between the Urea Cycle and the TCA Cycle
α keto acid
Aspartate
Ornithine
urea
Citrulline
Arginine
α amino acid
argininosuccinate
Fumarate
H2O
fumerase
Transaminase
Aspartate
Urea
Cycle
Oxaloacetate
Citrate
Malate
Malate
TCA Cycle
Fumarate
-Ketoglutarate
Succinyl CoA
12. How is the urea cycle regulated?
a. Long term regulation:
i. Increased metabolism of AAs  Increased synthesis of Urea cycle enzymes
1. Due to high protein diet (Carbon skeletons are oxidized and converted
to fat or glycogen)
2. Due to starvation (Carbon skeletons from protein catabolism for energy
are oxidized)
b. Short term regulation:
i. Arginine activates N-acetylglutamate (signals that high levels of AAs present)
ii. Acetyl-CoA + glutamate  N-acetylglutamate + CoA
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Amino Acid Metabolism: Objectives
13. Explain why ammonia is toxic. Explain the elements of inter-organ metabolism that contribute
to the self-perpetuating nature of ammonia intoxication.
a. Ammonia is toxic, because when its concentration is high…
i. Stimulates glucagon release from the pancreas
1. Which stimulates gluconeogenesis from AAs in the kidneys
a. Results in even higher ammonium level
i. Further stimulating glucagon release
1. Stimulate release of glucose (gluconeogenesis)
a. Promotes insulin release
i. Stim uptake of branched chain
AAS by muscle
b. Muscle uses AAs as oxidizable
substrates
2. That metabolism causes release of more
ammonium ions
ii. In the brain, stimulates glutamine synthesis, promotes
tryptophan transport
b. Increase of tryptophan, increase the synthesis of 5-OHtryptamine (an important neurotransmitter)
2. Causing depletion of alpha-ketogluterate and glutamate in brain!
b. It obviously has a cascading effect! Higher ammonia []causes even higher ammonia []!
c. Impaired Mitochondrial Function with Ammonia Intoxication
i. Increased Ca++ uptake, causing the pump to work harder (which requires ATP)
ii. Decrease ETC activity, and thus the ATP production is slowed
iii. Increased formation of free radicals and also NO
iv. Increase in lipid peroxidation
14. What TCA cycle intermediate is depleted in the brain during ammonia intoxication and how
does this happen?
i. Alpha-ketogluterate is the TCA intermediate which is depleted in the brain
ii. How it happens: See #13 for the cascade of happenings
b. What amino acids are depleted in the brain?
i. Alpha-ketogluterate
ii. Glutamate
c. What are the consequences of depletion of these metabolites for the brain?
i. Inhibits TCA cycle and thus lowers ATP production, BUT increase in ATP need
15. How is ammonia intoxication treated and how do these treatments work?
a. Diet: Low protein, high carb (which will reduce ammonia production)
b. Levulose: Bacteria utilize this in colon a more acidic env.  excrete NH4+ in the feces
c. Antibiotics: Kills intestinal ammonia-producing bacteria in the gut
d. Sodium Benzoate and Sodium Phenylbutyrate: form covalent products with glycine and
glutamine; promoting N excretion in the feces
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Amino Acid Metabolism: Objectives
16. Explain the distinction between a ketogenic vs a gluconeogenic fate of an amino acid. Which
amino acids are exclusively ketogenic and to what are they ultimately metabolized?
a. Ketogenic fate: Yield precursors which go to either fatty acids or ketone bodies
i. Amino acids which are metabolized to Acetyl-CoA or Acetoacetyl-CoA
ii. Amino acids which are exclusively ketogenic include:
1. Leucine: goes to Acetyl-CoA
2. Lysine: goes to alpha-ketoadipate, which goes to Acetyl-CoA
b. Gluconeogenic fate AKA Glucogenic fate: amino acids which yield precursors that can be
used for gluconeogenesis (can produce glucose)
i. Metabolize to:
1. Pyruvate
a. Include: Alanine, Cysteine, Serine, Threonine, Glycine
2. Alpha-ketogluterate
a. Include: Arginine, Histidine, Glutamine, Proline
3. Succinyl-CoA
a. Include: Methionine, Valine
4. Fumarate
a. Include: Tyrosine
5. Oxaloacetate
a. Include: Asparagine, Asparatate
c. Both Ketogenic and glucogenic fates are possible
i. Isoleucine:
Acetyl CoA
Propionyl CoA (Succinyl CoA)
ii. Tyrosine:
Acetoacetate
Fumarate
iii. Phenylalanine:
Acetoacetate
Fumarate
1. –forms into tyrosine
iv. Tryptophan:
Acetyl CoA
Others…
17. To what TCA cycle intermediates are individual amino acids metabolized and where do these
metabolites enter the TCA cycle.
Alanine
Serine
Glycine
Threonine
Cysteine
Tryptophan
(through alanine)
CO2
Pyruvate
Acetoacetate
Acetyl CoA
Oxaloacetate
Phenylalanine
Tyrosine
Tryptophan
Lysine
Citrate
Asparagine
Aspartate
CO2
Fumarate
Tyrosine
Phenylalanine
Aspartate
Isoleucine
Methionine
Valine
-Ketoglutarate
Glutamate
Succinyl CoA
Methylmalonyl CoA
CO2
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Glutamine
Proline
Arginine
Histidine
Amino Acid Metabolism: Objectives
18. What metabolites accumulate in individuals with a defect in the branched chain keto acid
dehydrogenase? What amino acids are metabolized by the pathway involving this enzyme?
a. Branched Chain Keto Aciduria (AKA Maple Syrup Disease)
i. Metabolites which accumulate:
1. Alpha-ketoiscaproate, alpha-keto-beta-methylvalerate, and
alpha-ketoisovalerate
ii. Enzyme with the defect is alpha-ketoacid(branched chain) dehydrogenase
1. AKA: BCKAD
iii. Amino acids which are metabolized by the pathway involving BCKAD include:
1. Leucine, Valine, Isoleucine
iv. Manifestation: severe mental retardation
1. Rare, recessive mutation isolated in Asian and Mennonite communities
19. What metabolites accumulate in alkaptonuria and what enzyme is defective in this disease?
a. Accumulating metabolites include:
i. Homogentisate
1. Causes urine to turn black on reaction with O2, at an alkaline pH
ii. Phenylalanine
iii. Tyrosine
b. Enzyme: Homogentisate Oxidase
i. Homogentisate  Maleylacetoacetic acid
c. Manifestation: damage to joints (arthritis), calcifications in the CVS, reddish tint (ochre)
to the skin
20. How is S-adenosyl methionine synthesized?
Synthesis of S-adenosyl Methionine
S-adenosylmethionine (SAM) is an
important methyl group donor.
Methionine
adenosyltransferase
+ ATP
PPi + Pi
S-adenosylmethionine
(SAM or Adomet)
methionine
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Amino Acid Metabolism: Objectives
21. How is methionine regenerated from homocysteine? What coenzyme is involved in this
reaction? What other enzyme catalyzed reaction in mammals utilized this coenzyme?
a.
N5-CH3 THF
THF
Methionine
Synthase
(B12 enzyme)
CH3
group
Methionine
Homocysteine
Adenosine
SAM methyl group donor in many reactions; e.g.:
Norepinephrine
epinephrine
Cytosine methylation of DNA
b. Coenzyme: B12
c. B12 is also needed for Methylmalonyl CoA  Succinyl CoS (along with the enzyme:
Methylmalonyl-CoA Mutase)
22. What enzyme is defective in individuals with homocystinuria? What metabolite accumulates?
How is this disease usually treated?
a. Defective enzyme: Cysathionine Beta-Synthase
i. Which is a vitamin B6 (pyridoxal phosphate) dependent enzyme
b. Accumulating Metabolite:
i. Homocysteine
c. Treatment:
i. If Vitamin responsive: treat with Vit B6 (pyridoxine), folic acid, vit B12
ii. If Vitamin Unresponsive: Restrict methionine
1. Supplement with cystine and betaine
a. Promotes re-methylation of homocysteine to methionine
23. How are homocysteine thiolacetone and S-nitrosohomocysteine formed?
a. They are formed from homocysteine’s reaction with protein amino groups
i. Makes the modified protein immunogenic
b. S-nitrosohomocysteine is formed from homocysteine and NO
c. Homocysteine thiolacetone is formed from homocysteine
i. By the enzyme: Methionyl-tRNA Synthetase
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Amino Acid Metabolism: Objectives
24. Which amino acids can be made from intermediates of a central pathway and what
intermediate gives rise to which amino acids?
a. AAs made from intermediates of a central pathway:
i. Transamination from a keto acid:
1. Pyruvate alanine
2. Alpha-ketogluterate  glutamate
3. Oxaloacetate  aspartate
ii. Transamination of a keto acid followed by amidation:
1. Alpha-ketogluterate  glutamate  glutamine
2. Oxaloacetate  aspartate  Asparagine
iii. Reductive Amidation:
1. Alpha-ketogluterate  Glutamate (Enzyme: Glutamate Dhd)
iv. From other intermediates of a central metabolic pathway:
1. Phosphoglycerate  Serine
2. Phosphoglycerate  Serine  Glycine
3. Citrulline  Argininorsuccinate  Arginine
v. From other amino acids:
1. Phenylalanine  Tyrosine
2. Glutamate  Proline
3. Methionine  Cysteine
25. Explain the role of the small intestine and kidney in the synthesis of arginine.
a. Synthesis of arginine begins in the enterocytes of the small intestine
i. Glutamine y-semialdehyde + Glutamate  Ornithine  Citrulline
ii. Citrulline is carried by the circulation to the kidney
b. Synthesis of arginine continues in the kidney
i. Citrulline+ Aspartate + ATP  Argininosuccinate  Fumerate + Arginine
ii. Enzymes: 1st – argininosuccinate synthetase 2nd – argininosuccinate lyase
c. The kidney lacks arginase of the urea cycle
i. Thus, the kidney is a major site of arginine production
d. This pathway is AKA “Intestinal-Renal Axis”
26. What reaction is catalyzed by phenylalanine hydroxylase and what is the role of
tetrahydrobiopterin in this reaction. What is phenylketonuria?
a. Phenylalanine  Tyrosine
Enzyme: Phenylalanine hydroxylase
b. Tetrahydrobiopterin is the substrate (along with phenylalanine) for the formation of
tyrosine
i. Needs to be produced from dihydro-biopterin + NADH
1. Enzyme: Dihydropteridine Reductase
c. Phenylketonuria: Defect in a phenylalanine hydroxylase gene
i. Occasionally, this defect involves the dihydrobiopterin cofactor
ii. Tx: Supplement with tyrosine, avoid phenylalanine
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Amino Acid Metabolism: Objectives
27. How is nitric oxide made? What are some of the functions of this short-lived molecule?
a. Arginine + O2 + NADPH  Citrulline + NADP+ + NO
b. Functions:
i. iNOS (AKA NOSI): Immune and inflammatory responses
ii. nNOS (AKA NOSII): Neurotransmitter in the GI tract, in penile erection, and in
sphincter relaxation
- acts on smooth muscle
iii. eNOS (AKA NOSIII): Acts on endothelial cells; blood flow, blood pressure, and
platelet activation
Citrulline Nitric
oxide Cycle
ATP
AMP + PPi
Citrulline
ASS
Aspartate
Citrulline
NO
Argininosuccinate
NOS
Arginine
ASL
Fumarate
Just say NO.
O2 ASS – Argininosuccinate synthetase
c.
ASL – Argininosuccinate lyase
NOS – Nitric oxide synthase
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