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December 7-8, 2011

AMINO ACID METABOLISM I,II,III Lecturer: Eileen M. Lafer

Reading: Stryer Edition 6: Chapters 23 and 24. Figures in this document are from
Stryer unless otherwise noted.

OBJECTIVES:

1. Understand the fates and sources of the amino acids in general terms.

2. Understand the general features of lysosomal protein degradation, including what


types of proteins are degraded in lysosomes.

3. Understand how "controlled proteolysis" is effected in cells. This includes an


understanding of the mechanism of selection and attachment of ubiquitin to target
proteins, as well as where and how ubiquitinated proteins are degraded.

4. Understand the common pathways for the removal of the -amino group from an -
amino acid during amino acid catabolism.

5. Understand how the fate of the ammonium ions generated during amino acid
degradation differs in the liver versus the peripheral tissues.

6. Understand the fundamentals of the urea cycle and how it is regulated. Understand
how defective urea cycle enzymes can lead to disease, and how those diseases are
treated.

7. Understand which -amino acid carbon skeletons feed into which major metabolic
intermediates during amino acid catabolism.

8. Know which amino acids are solely ketogenic, solely glucogenic, and both ketogenic
and glucogenic.

9. Know which steps in amino acid degradation lead to the following diseases: methyl-
malonic acedemia, homocystinuria, maple syrup disease, phenylketonuria, tyrosinemia
I, II and III, and alkaptonuria. For each disease, you should know the name of the
defective enzyme, the reaction catalyzed by the enzyme, and the pathway in which the
enzyme functions.

10. Know which amino acids are essential and which are non-essential in humans.

11. Know in humans, which major metabolic intermediates are able to serve as carbon
skeletons for the biosynthesis of which amino acids.

12. Know which amino acids can act as neurotransmitters.

13. Know which amino acids can be used for the synthesis of which neurotransmitters.

14. Understand in general terms the biosynthesis of spermine, spermidine, creatine and
phosphocreatine.

Reminder: to download movies used in my lectures go to my web page:


http://www.biochem.uthscsa.edu/~lafer/
(alternatively go to UTHSCSA biochem dept., click on faculty, etc. to find above link)
Click on links, for a mirror of the ppt files on blackboard, as well as the movie files that
are not permitted on blackboard due to size limitations.

A. SOURCES AND FATES OF AMINO ACIDS IN THE BODY

I. SOURCES OF AMINO ACIDS IN THE HUMAN BODY: DIETARY


PROTEIN, BIOSYNTHESIS AND TISSUE BREAKDOWN.

II. FATES OF AMINO ACIDS IN THE HUMAN BODY: EXCRETION,


ENERGY METABOLISM, AND BIOSYNTHESIS OF SMALL BIOMOLECULES.

B. PROTEIN CATABOLISM

I. DIET Proteolysis of Proteolysis of Controlled


dietary proteins proteins that proteolysis of
1. ~1/3 in the stomach move through ubiquitin-tagged
of the amino acids in
and lumen of the the endocytic intracellular
the amino acid pool
come from dietary small intestine pathway takes proteins takes
proteins. releases free place in the place in the
2. ~2/3 amino acids into lysosomes of proteasomes of
of the amino acids in the bloodstream. all cells. all cells.
the amino acid pool
come from
intracellular sources.
AMINO ACID POOL

II. LYSOSOMAL DEGRADATION

1. Lysosomes degrade proteins taken up by endocytosis, or proteins that


traffic within the endocytic pathway.
2. Lysosomes contain ~50 hydrolytic enzymes (proteases). Their pH
optima is acidic.
3. The pH of the lysosome is ~5.
4. In well-nourished cells, lysosomal protein degradation is non-selective.
5. In starving cells, there is a
selective pathway that preferentially degrades The Mark of Death
cytosolic proteins containing the pentapeptide
KFERQ (Lys-Phe-Glu-Arg-Gln).

III. CONTROLLED PROTEOLYSIS

1. Ubiquitin tags proteins for


destruction.
2. The proteasome digests the
ubiquitin tagged proteins.
3. Protein degradation can be used
to regulate biological function.
4. Properties of Ubiquitin:
a. 76 aa polypeptide.
b. C-terminal gly attaches to
the -amino groups of several lys on a protein
destined for degradation.
c. Additional ubiquitin
molecules can be added to Lys48.

5. Ubiquitin Conjugation: E1 adenylates ubiquitin , and transfers it to its


own cysteine. Ubiquitin is then transferred to a cysteine in E2. Finally E3 catalyzes the
final transfer to a lysine residue on the target protein.

E1=Ubiquitin-Activating
Enzyme
E2=Ubiquitin-
Conjugating Enzyme
E3=Ubiquitin-Protein
Ligase

6.
Ubiquitin is attached to
the -amino group of
lysine residues on
target proteins.

7. Clinical correlation: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 all
cervical carcinomas.

8. A single ubiquitin molecule is a poor signal for degradation. However,


chains of 4 or more ubiquitin molecules are very strong signals for degradation.

9. What determines whether a


protein is ubiquitinated? The substrate
specificity of each E3:

a. The N-terminal rule: the


chemical nature of the amino-terminal amino
acid. For example, a protein with methionine
at its N terminus has a half life of 20 hours,
while a protein with an arginine at its N-
terminus has a half life of 2 minutes.
.
b. Cyclin destructive
boxes: specific amino acid sequences that
mark cell-cycle proteins for destruction.

c. Pest sequences: proteins rich in proline, glutamic acid, serine


and threonine.

10. The 26s proteasome digests the ubiquitin tagged proteins.

19S regulatory subunit

20S proteasome

(catalytic activity)

19S regulatory subunit

11. The 20s proteasome

a. 700kD,
28 homologous
subunits: 14 of
type and 14 of
type .

b. Sub-
units are
arranged in 4
rings of 7 subunits each to form a sealed barrel.

12. The proteolytic activity resides in the N-terminal threonine residues of


the beta subunits.

a. Access to the 20s proteasome is controlled by the 19s caps.

The 19S regulatory subunits

bind to polyubiquitin chains.

IV. BREAKDOWN OF TISSUE PROTEINS

1. Protein degradation can regulate biological processes

Dynamically alter the stability of regulatory proteins

V. AMINO ACID DEGRADATION

1. Any amino acids generated by protein catabolism that are not needed
as building blocks for new biomolecular synthetic reactions are degraded to
carbon skeletons in the liver.

2. The first step in amino acid degradation is the removal of nitrogen.

-AMINO GROUPS ARE CONVERTED INTO AMMONIUM IONS BY OXIDATIVE


DEAMINATION OF GLUTAMATE

The -amino group of the


-amino acid is transferred

to -ketoglutarate to form
glutamate, which is
oxidatively deaminated to
yield ammonium ion.

a. THE TRANSAMINATION REACTION: Aminotransferases (also


called transaminases) catalyze the transfer of an -amino group from an
-amino acid to an -keto acid.

These enzymes generally utilize -ketoglutarate as the acceptor.

The enzymes are named after their amino acid substrates, i.e. aspartate transaminase
catalyzes the transfer of the -amino group of aspartate to -ketoglutarate, yielding
oxaloacetate plus glutamate.

b. THE OXIDATIVE DEAMINATION REACTION: The nitrogen


atom that is transferred to -ketoglutarate in the transamination reaction is
converted into free ammonium ion by oxidative deamination.

This reaction is catalyzed by glutamate dehydrogenase. This reaction


takes place in the mitochondria, and is driven by the consumption of
ammonia.

Dehydrogenation Hydrolysis

c. The sum of the aminotransferase and glutamate dehydrogenase


reactions yield ammonium ion:

aminotransferase dehydrogenase urea cycle

AMINO ACID 1 ALPHA KETOACID 2

excreted
ALPHA KETOACID 1 AMINO ACID 2

d. All aminotransferases
contain the prosthetic
group pyridoxal
phosphate (PLP).
PLP is derived from
Pyridoxine (Vitamin B6).

e. PLP Tautomers:
The phenolic hydroxyl group is slightly
acidic, favoring deprotonation.

The pyridine ring is slightly basic, which favors protonation of the pyrimidine N.

f. PLP forms Schiff base


intermediates in aminotransferases
AMINO ACID 1


(schiff-base linkage with the enzyme) (schiff-base linkage with the substrate)

The positively charged schiff-base linkages are


stabilized by the negatively charged phenolate group.

1. The schiff base loses a proton from the -carbon of the


amino acid to become a quinonoid intermediate.
2. Reprontonation of the quinonoid at the aldehyde carbon
yields a ketimine intermediate.
3. The ketimine is then hydrolyzed to an -ketoacid and
PMP.

ALPHA KETOACID 1

1 2 3

4. Once the amino group has been transferred to PMP,


PMP transfers the amino group to another alpha-ketoacid by reversing the reaction
scheme we ALPHA KETOACID 2
(ALPHA KETOGLUTARATE)
just discussed
(follow the red
arrows):

3 2 1

g
.
4

AMINO ACID 2
(GLUTAMATE)

g. The sum of the aminotransferase and glutamate


dehydrogenase reactions yield ammonium ion.

aminotransferase dehydrogenase urea cycle

AMINO ACID 1 ALPHA KETOACID 2

excreted
ALPHA KETOACID 1 AMINO ACID 2

h. Aspartate Aminotransferase

i. Mechanism of the Aminotransferase Reaction:

MOVIE: Movie file 18-01.avi

j. Serine and Threonine can be directly


deaminated

1. The nitrogen atoms of MOST amino


acids are transferred to -ketoglutarate.

2. The -amino groups of serine and


threonine can be directly converted into ammonium ion by the action
of dehydratases: Threonine -ketobutyrate + NH4+

k. Peripheral tissues transport nitrogen to the


liver by the alanine cycle or as glutamine.

If amino acids are produced in tissues that lack the urea cycle, they
need a mechanism to release nitrogen in a form that can be
absorbed by the liver and converted into urea.

EXAMPLE: Muscle uses amino acids as fuel during prolonged


exercise and fasting.

1. The Alanine Cycle

a. In peripheral tissues, the -


amino groups of the amino acids are transferred to glutamate by a transamination
reaction, as in the liver.

b. However, rather than oxidatively deaminating


glutamate to form ammonium ion, the amino group is transferred to pyruvate to form
alanine.

c. The liver takes up the alanine, and converts it back


to pyruvate by another transamination reaction.

d. The pyruvate can be used for gluconeogenesis,


and the amino group eventually ends up as urea by the usual pathway.

2 . Nitrogen can also be transported as glutamine:

Glutamine Synthetase:
NH4+ + glutamate + ATP


glutamine

+ ADP + Pi
a. Once glutamine is in the liver, it can be
metabolized like any other amino acid and the nitrogen can end up in the urea cycle.

IV. THE UREA CYCLE

1. In liver the ammonium ions generated during amino acid degradation


feed into the urea cycle.

2. Urea cycle: importance

a. NH4+ is a product of the breakdown of amino acids.

b. NH4+ is required by cells for synthesis of nitrogen-containing


compounds.

c. Excess NH4+ is very toxic. Normal levels in human blood are:


[NH4+] < 70 M.

d. Excess NH4+ is converted to urea via the urea cycle and


excreted. The urea cycle accounts of ~80% of the excreted nitrogen.

3. Urea cycle: location and source of atoms

a. Urea synthesis takes


place mostly in the liver.

b. One N atom of urea


comes from Asp (blue).

c. One N atom comes from


NH4+ (green).

d. One C atom comes from


CO2 (red).

e. Ornithine acts as a
carrier of various atoms
in the process of
synthesizing urea.

4. Urea cycle reactions: carbamoyl phosphate synthetase

a. Catalyzes formation of carbamoyl phosphate from H2O, 2


ATPs, CO2 and NH3.

b. The positive heterotropic activator, N-acetylglutamate, is


required for activity.

c. Brings one C atom and one N atom into the urea cycle as a
carbamoyl group.

d. Catalyzes the critical step in removing NH4+ from the blood.

5. Urea cycle reactions: Carbamoyl Phosphate synthetase

a. The reaction is made irreversible by cleaving two ATP


molecules to two ADP.

b. One molecule of phosphate is released while the second


phosphate ends up as part of carbamoyl phosphate.

c. Carbamoyl phosphate synthetase is present at very high


concentration in the mitochondrial matrix (~1 mM).

d. The high enzyme concentration allows the enzyme to work


well below the Km ~ 250 M for NH4+.

e. By operating well below Km, a small increase in NH4+ leads


to a large increase in the rate of removal of NH4+ insuring that NH4+ remains low.

6. Urea cycle reactions: ornithine transcarbamoylase

a. Catalyzes the formation of citrulline and Pi from ornithine and


carbamoyl phosphate.

b. Transfer of a carbamoyl group to ornithine is facilitated by


rupture of a high energy phosphoanhydride bond.

c. Catalyzes introduction of one C atom and one N atom into the


urea cycle from carbamoyl phosphate.

7. Urea cycle reactions: argininosuccinate synthetase

a. Catalyzes condensation of citrulline and aspartate to form


argininosuccinate.

b. Catalyzes the introduction of one N atom into the urea cycle


from aspartate.

8. Urea cycle reactions: argininosuccinase

a. Cleaves argininosuccinate to arginine and fumarate.

b. Completes the transfer of the amino group from aspartate to


make arginine.

c. Retains the carbon skeleton of aspartate (as a fumarate


molecule).

9. Urea cycle reactions: arginase

a. Catalyzes hydrolysis of arginine to ornithine and urea.

b. Ornithine cycles back to the first step and picks up another


carbamoyl group from carbamoyl phosphate.

10. Urea cycle: overall reaction

a. PPi 2 Pi quickly in a
reaction catalyzed by
pyrophosphotase.

b. Overall, four high energy
phosphate bonds are
broken to synthesize
each molecule of urea.


11. Urea cycle and the citric acid cycle

a. Fumarate production connects the urea cycle and the citric acid
cycle (fumarate malate oxaloacetate).

b. In the citric acid cycle fumarate is converted to oxaloacetate.

c. Oxaloacetate is transaminated to aspartate.

d. Aspartate carries the amino groups of other amino acids into


the urea cycle.

12. Compartmentalization of the urea cycle


a. Takes place in the liver.

b. Two intracellular

locations.

c. Mitochondrial matrix:
carbamoyl
phosphate
formation and citrulline

synthesis.

d. Cytosol:
argininosuccinate

formation; cleavage of
argininosuccinate
to
arginine and fumarate;
hydrolysis of arginine to
ornithine and urea.

13. Regulation of the urea cycle

a. The
urea cycle removes
excess NH4+ which
comes from the
breakdown of amino
acids.

b.
Overall control of the
urea cycle is by
enzyme levels, which
change by as much
as ten-fold depending
on the diet.

c. The flow of compounds through the urea cycle also depends on


the concentrations of cycle intermediates.

d. Several reactions convert amino acids into urea cycle


intermediates.

e. Arginine from the diet can be converted to ornithine.

f. Glutamate can be converted to ornithine by intestinal enzymes.

g. Fine control of the urea cycle is through regulation of carbamoyl


phosphate synthetase.

h. N-acetylglutamate is a heterotropic allosteric activator of


carbamoyl phosphate synthetase. (Heterotropic means an effector molecule that
is different from the substrate.)

i. N-acetylglutamate acts as a signal for high amino acid


concentrations.

j. N-acetylglutamate is synthesized in the liver from acetyl-CoA and


glutamate in a reaction catalyzed by N-acetylglutamate synthetase.

k. The steady state concentration of N-acetylglutamate is


determined by two factors: 1. Concentrations of substrates: acetyl CoA and
glutamate. 2. Concentration of arginine, which activates N-acetylglutamate
synthetase.

14. Defective urea cycle enzymes and inherited disease.

a. High NH4+ is toxic and a complete lack of any urea cycle


enzyme is fatal.

b. Some diseases are believed to be due to partially active


defective enzymes.

c. Defective enzymes cause high levels of NH4+ in the blood.

d. Sometimes a low protein diet can help. The diet decreases the
amount of NH4+ that needs to be eliminated through the urea cycle.

15. Defective enzymes and treatment

a. Problem: argininosuccinase deficiency.

1. Treatment: a low protein diet high in


arginine. NOTE THIS IS THE PRODUCT
OF THE DEFECTIVE ENZYME

2. Result: argininosuccinate is secreted in


place of urea to remove ammonium ions
that are generated during amino acid
catabolism.

b. Problem: carbamoyl phosphate synthetase or ornithine


transcarbamoylase deficiency.

1. Result: glycine and glutamine build up (pyruvate and


glutamate accept amino groups from ammonium ions).

2. Treatment: feed benzoate to remove Gly as hippurate.


Feed phenylacetate to remove Gln as phenylacetylglutamine

16. NH4+ toxicity and excess glutamine


a. NH4+ toxicity may be


due to formation of excess
glutamine.

b. High glutamine levels


are found in the
cerebrospinal fluid of those with high NH4+.

c. High glutamine and glutamate may lead to brain damage, possibly by producing
osmotic effects that cause brain swelling


VII. FATES OF THE CARBON SKELETONS OF THE AMINO ACIDS


All 20 amino acids funnel into only 7 major metabolic intermediates.

1. The strategy of

amino acid degradation is to


transform the carbon
skeletons
into major metabolic
intermediates that can be
converted into glucose,

or oxidized
by the citric acid cycle.

2. The carbon
skeletons of a diverse
set of 20
amino acids are funneled into only

7 molecules: pyruvate, acetyl
CoA, acetoacetyl CoA, -
ketoglutarate, succinyl CoA,
fumarate and oxaloacetate.

a. Pyruvate as an entry point into metabolism


3carbonaminoacids
ala,ser,cysentervia

pyruvate.

b. Oxaloacetate as an entry point


into metabolism

1. The skeletons of 4-
carbon amino acids enter at oxaloacetate.

(Figure from Principles of Biochemistry by Lehninger


and Fox, Freeman Publishing)

c. -Ketoglutarate as an entry
point into metabolism

1. The skeletons of several 5-carbon amino acids enter the


TCA cycle at -ketoglutarate. All first transfer their amino groups to glutamate, which is
then oxidatively deaminated by glutamate dehydrogenase to yield -ketoglutarate.

d. Succinyl Coenzyme aa as an entry point into metabolism for


several non-polar amino acids

e. Clinical
Correlations:

1. Homocystinuria:
Scoliosis, muscle
weakness, mental
retardation, thin
blond hair
Cystathione -
synthase is defective.

2. Methyl-malonic
academia:
Methylmalonyl- CoA
mutase is defective.

(Figure from
Principles of
Biochemistry by
Lehninger and Fox,
Freeman Publishing)

f. Ketogenic versus glucogenic amino acids

3. Metabolism of the branched chain amino acids.

a. When the branched chain amino acids valine, isoleucine, and


leucine are degraded in extra-hepatic tissues they share two common enzymes:

branched-chain aminotransferase

branched-chain -ketoacid dehydrogenase


complex

b. While much of the catabolism of amino acids takes place in the


liver, the branched chain amino acids are oxidized as primary fuels in muscle, adipose,
kidney, and brain tissues.

c. CLINICAL CORRELATION: maple syrup disease; Defective


branched-chain -keto acid dehydrogenase complex; urine has odor of maple syrup,
mental and physical retardation UNLESS patients are placed on a diet low in valine,
isoleucine and leucine early in life. (Figure from Principles of Biochemistry by
Lehninger and Fox, Freeman Publishing)

4. Oxygenases are required for the degradation of aromatic amino acids.

a. Monooxygenase- one atom of O2 appears in the product and


one in H20.

b. Clinical Correlation: Mutations in the gene encoding


phenylalanine hydroxylase cause Phenylketonuria

More than 200 mutations have been


identified. Mutations effecting the active
site, the biopterin binding site, and other
regions of the protein are indicated as
colored spheres.





c. Clinical Correlation:
In addition to PKU, a number of other
diseases are caused by defects in aromatic
amino acid metabolism. (Figure from
Principles of Biochemistry by Lehninger and
Fox, Freeman Publishing)

Phenylketonuria-phenylalanine
accumulates in body fluids, if untreated,
severe mental retardation. Treatment-

low phenylalanine diet.

Tyrosinemias-if untreated, weakness,


self-mutilation, liver damage, mental
retardation. Treatment?

Alkaptonuria-homogentisate
accumulates in the urine, and is
excreted, which turns dark on
standing due to the oxidation of

homogentisate.

Harmless

(Figure from Principles of


Biochemistry by Lehninger and
Fox, Freeman Publishing)

d. Tryptophan
Degradation

1. Nearly all
cleavages of aromatic rings in biological systems are catalyzed by dioxygenases.

e. Summary of defects in Amino Acid Catabolism that contribute to


human disease. (Table from Principles of Biochemistry by Lehninger and Fox,
Freeman Publishing)

C. AMINO ACID BIOSYNTHESIS

I. THE NITROGEN IN AMINO ACIDS, PURINES, PYRIMIDINES AND OTHER


BIOMOLECULES ULTIMATELY COMES FROM ATMOSPHERIC N2.

II. THIS PROCESS BEGINS WITH THE REDUCTION OF N2 TO NH3. THIS


PROCESS IS CALLED NITROGEN FIXATION, AND IS CARRIED OUT BY SOME
BACTERIA.

III. SINCE NITROGEN FIXATION DOES NOT TAKE PLACE IN HIGHER


ORGANISMS, THE SOURCE OF NITROGEN FOR AMINO ACID BIOSYNTHESIS IN
HUMANS ARE THE METABOLITES OF DIETARY NITROGEN.

IV. AMINO ACIDS ARE MADE FROM METABOLITES OF THE MAJOR


METABOLIC PATHWAYS.

V . MOST MICROORGANISMS CAN SYNTHESIZE ALL 20 AMINO ACIDS


HUMANS CAN ONLY SYNTHESIZE 11 AMINO ACIDS

1. The synthesis of many of


the amino acids is a simple reversal of
their degradation, utilizing a
transamination reaction.

2. Amino acids skeletons end


up as major metabolic intermediates
during degradation. Likewise, amino acids
are also biosynthesized from major
metabolic intermediates.

D. BIOSYNTHETIC FATES OF THE AMINO ACIDS:

I. NEUROTRANSMITTER BIOSYNTHESIS (Figures in the sections that follow are


from Principles of Biochemistry by Lehninger and Fox, Freeman Publishing)

Several amino acids can function as neurotransmitters without any chemical


modification: Glutamate, Glycine, Aspartate

Most of the small-molecule neurotransmitters are amino acids or their derivatives

Tyrosine
*


Tyrosine


Tyrosine


Glutamate
Tryptophan

GABA
Histidine

* (not directly from an amino acid, from acetyl CoA + choline



1. Catecholamine Biosynthesis 3. Histamine Biosynthesis

4. Serotonin Biosynthesis

2. GABA Biosynthesis

II. BIOGENIC AMINE BIOSYNTHESIS

III. BIOSYNTHESIS OF CREATINE AND PHOSPHOCREATINE

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