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Chapter 20: Amino acid metabolism

Takusagawas Note

Chapter 20: Amino Acid Metabolism

Amino acids from proteins are:
- precursors of compounds
- energy source (i.e., converted to acetyl-CoA, etc.)
Amino acids are obtained in diet and/or turnover of cellular proteins.
Major problem in amino acid degradation is elimination of amino group (-NH2) since NH3 from
-NH2 is very toxic.
Ammonia eliminations are:
- Conversion to urea (mammals)
- Conversion to uric acid (birds)
Carbon skeleton of amino acid metabolism is:
- NH2 group is removed by transamination & oxidative deamination to urea.
Transamination
-

COO

O
-

R1

E-PLP

-KA1

R CH C O
L-amino acid

COO

R1 C COO + R2 CH NH2

CH NH2 + R2 C COO
AA1
-KA2

H3N+

C CH2 CH2 C C O
-Ketoglutarate

H3N

R C
C O
-Keto acid

CH2 CH C O
Aspartate

transaminase

O
-

H3N

transaminase
O

AA2

CH2 CH2 CH C O
Glutamate

CH2 C C O
Oxaloacetate

O
-

Then Asp Urea

Oxidative deamination

H3N+

R CH C O
L-amino acid

O
-

C CH2 CH2 C C O
-ketoglutarate NH3

transaminase
O

R C
C O
-keto acid

H3N+ O

O
-

C CH2 CH2 CH C O
glutamate
Urea

NADH + H+
glutamate
dehydrogenase
+
NAD + H2O

Chapter 20: Amino acid metabolism

Takusagawas Note

Pyridoxal-5-phosphate (PLP) is co-enzyme (co-factor) of transaminase.

Aminotransferase reactions occur in two stages (Ping-Pong Bi Bi reaction):

1. Amino acid + Enzyme -Keto acid + Enzyme-NH2
2. -Ketoglutarate + Enzyme-NH2 Enzyme + Glutamate
Details of aminotransferase reactions are shown in Fig. 24-2.
Stage-0: Enzyme-PLP Schiff base formation
PLP is covalently attached to the enzyme via a Schiff base linkage between aldehyde group
of PLP and Lys (-amino group) of enzyme.
E-Lys + PLP E-PLP

(CH2)4
H
2-

O3PO

H2
C

H
-

O
+
N
H

CH3

Enzyme

Chapter 20: Amino acid metabolism

Takusagawas Note

Stage I: Conversion of an amino acid to -keto Acid

1. Transimination: Amino acids nucleophilic amino group attacks the E-PLP Schiff base
carbon atom in a transimination reaction to form E-PLP-AA. Then E-PLP-AA is E-Lys +
PLP-AA.
E-PLP + AA [E-PLP-AA] E-Lys + PLP-AA

2. Tautomerization: AA-PLP tautomerizes to an -keto acid-PMP by the active-site Lys

catalyzed removal of the amino acid -hydrogen and protonation of PLP atom C4.
AA-PLP -Keto acid-PMP

Chapter 20: Amino acid metabolism

Takusagawas Note

3. Hydrolysis: -Keto acid-PMP is hydrolyzed to PMP and -Keto acid.

-Keto acid-PMP + H2O -Keto acid + PMP

Stage II: Conversion of an -keto acid to an amino acid (reverse reactions of stage I)
3. -Keto acid + PMP -Keto acid-PMP
2. -Keto acid-PMP AA-PLP
1. E-Lys + PLP-AA [E-PLP-AA] E-PLP + AA
Note: All amino acids form the E-PLP-AA intermediate: AA + E-PLP E-PLP-AA.
3

E-PLP-AA is then converted by:

1. Transamination
2. Decarboxylation
3. Elimination from - or -carbon
4. Racemization (D L)
5. Others

4
H

C C COO

HN
1
-

HC

OH

O3P O
N
H

NH2 E

CH3

Takusagawas Note

Chapter 20: Amino acid metabolism

Urea Cycle
Urea is formed from ammonia (NH3), amino group (NH2) of Asp, and bicarbonate (HCO3-) by
urea cycle in liver.
O
H2N

HCO3

NH2

NH3

NH2 of Asp

Five enzymes are involved in urea synthesis in urea cycle.

Two enzymes are in mitochondrion.
Three enzymes are in cytosol.
Therefore, the urea cycle occurs partially in the mitochondrion and partially in the cytosol.

Chapter 20: Amino acid metabolism

Takusagawas Note

Reactions in urea cycle

1. Carbamoyl phosphate synthetase (Regulating enzyme)
Formation of carbamoyl phosphate from NH3 and HCO3- (bicarbonate) using ATP as energy
source.
HCO3- + NH3 + 2ATP H2N-CO(OPO32-) + 2ADP + Pi

1st ATP

2nd ATP

2. Ornithine transcarbamoylase
Transfer carbamoyl group (O=C-NH2) to ornithine to produce citrulline.
Ornithine + O=C-NH2(PO32-) Citrulline + Pi
3. Argininosuccinate synthetase
Acquisition of the second urea nitrogen atom from Asp.
Citrulline + Asp Argininosuccinate

Chapter 20: Amino acid metabolism

Takusagawas Note

4. Argininosuccinase
Elimination of arginine from the aspartate carbon skeleton to form fumarate.
Argininosuccinate Fumarate + Arginine
5. Arginase
Hydrolysis of arginine to yield urea and regenerate ornithine.
Arginine Urea + Ornithine

Overall reaction of urea cycle is:

CO2 + NH3+ + 3ATP + Asp + 2H2O Urea + 2ADP + 2Pi + AMP + PPi ( 2Pi) +
Fumarate
The urea cycle converts two amino groups (one from NH3 and one from Asp) and a carbon atom
(HCO3-) to non-toxic excretion product, urea, at the cost of 4 high-energy phosphate bonds
(i.e., 4ATP).
However, oxidations of urea cycles substrate (Glu) and product (malate) produce 2 NADH (= 6
ATP) as shown in Fig. 24-7.

Chapter 20: Amino acid metabolism

Takusagawas Note

The urea cycle is conjunct with apartate-argininosuccinate shunt of tricarboxylic acid (TCA)
cycle as shown below. This is called Krebs bicycle. Note: tricarboxylic acid cycle =
citric acid cycle = Krebs cycle.

Oxaloacetate is one of the most important precursor of:

CAC (condenses with acetyl-CoA)

Oxaloacetate

Gluconeogenesis
Asp

Urea
Protein

Regulation of the urea cycle

- is regulated by carbamoyl phosphate synthetase.
- Carbamoyl phosphate synthetase is allosterically activated by N-acetylglutamate. Thus, Nacetyl-glutamate plays an important role in urea cycle regulation.

COO(CH2)2 O
H C N C CH3
H
OOC
8
N-Acetyl-glutamate

Chapter 20: Amino acid metabolism

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Takusagawas Note

Acetyl-Glu is synthesized by acetyl-glutamate synthase

Glu + Acetyl-CoA N-acetyl-Glu.
N-acetyl-Glu formation can be as follows:
1. Breakdown of protein produces amino acids including Glu (i.e., [Glu] ).
2. Need urea cycle to be activated since amino acid degradation produces amines.
3. In the mean time, [Glu] causes [N-acetyl-Glu]
4. [N-acetyl-Glu] increases the activity of carbamoyl phosphate synthetase. Thus, urea
cycle is activated.

Ammonia transport mechanism

- Ammonia (NH3) is produced in all tissue, but the urea cycle is only carried out in liver.
Thus, NH3 must be transported to liver with non-toxic form.
NH3 is converted to glutamine (Gln) which is not toxic.
Glutamine synthetase
ATP + NH4+ + Glu
ADP + Pi + Gln + H+
- Gln is hydrolyzed to Glu and NH4 in liver.
glutaminase
Gln + H2O Glu + NH4+
- NH4+ is converted to urea.
Another special system between muscle and liver to get nitrogen to the liver: Glucose-alanine
cycle is shown below.
- Amino group in Glu produced from amino acids NH3 in muscle is transferred to pyruvate.
- The aminated pyruvate, Ala, is transported to liver where the NH2 is transported to ketoglutarate.
- The aminated -ketoglutarate, Glu, releases NH3.
- NH3 enters the urea cycle and is converted to urea.
- In this glucose-alanine cycle, muscle uses glucose and excretes nitrogen, whereas liver
converts alanine to glucose and excretes NH3 in urea cycle.

Chapter 20: Amino acid metabolism

Takusagawas Note

10

Amino acids skeleton metabolism

Keto
Leu
Lys

Keto &
Gluco
Ile
Thr
Phe
Try
Trp

Gluco
Ala
Cys
Gly
Ser
Asp
Asn
Met
Val
Arg
Glu
Gln
His
Pro

20 amino acids are converted to 7 common intermediates. Those are:

1. Pyruvate
2.
3.
4.
5.

-ketoglutarate
Succinyl-CoA
Fumarate
Oxaloacetate

6. Acetyl-CoA
7. Acetoacetate

Both glucogenic and ketogenic intermediate (do not confuse!)

Glucogenic intermediates (form glucose)

Ketogenic intermediates (form ketone bodies)

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Chapter 20: Amino acid metabolism

11

Takusagawas Note

Example of Amino acid degradation

Alanine, Cysteine, Glycine, Serine, and Threonine are degraded to Pyruvate
- Degradations of these amino acids involve:
1. Elimination of -NH2, -OH, -SH
2. Transfer of hydroxymethyl group
3. Oxidation-reduction
- Pathways are shown Fig. 24-9.

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Chapter 20: Amino acid metabolism

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Takusagawas Note

Amino acid biosynthesis

Tetrahydrofolate Cofactors: Metabolism of C1 Units
- Tetrahydrofolate (THF) functions to transfer C1 units in several oxidation states.
- Most reactions require NADPH/NADH.
- THF is composed of three units:
2-Amino-4-oxo-6-methylpterin
p-Aminobenzoic acid
Glutamates

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Chapter 20: Amino acid metabolism

-

13

Takusagawas Note

THF is derived from folic acid (one of vitamin) by two-stage reduction. Both reactions are
catalyzed by dihydrofolate reductase (DHFR).
Inhibition of DHFR inhibits nucleic acid synthesis since THF transfers C1 units to
biosyntheses of proteins and nucleic acids.

N5 and N10 in THF are important nitrogens, since C1 units are covalently attached to THF at
its positions 5N, 10N, or both 5N and 10N.
C1 units are listed in Table 1.

The C1 units carried by THF are interconverted to:

Methionine
Thymidylate (dTMP)
Formylmethionine-tRNA
Purines

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Chapter 20: Amino acid metabolism

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Takusagawas Note

Reactions involved in THF are oxidation-reduction, cyclization and hydrolysis

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Takusagawas Note

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Chapter 20: Amino acid metabolism

Sulfonamides competitively inhibit bacterial synthesis of THF

O
H2N

S NH R

O
Sulfonamides
(R = H sulfanilamide)

O
H2N

C OH

p-Aminobenzoic acid

Why? Because sulfonamides are:

- structural analogs of p-aminobenzoic acid constituent of THF.
- antibiotics (sulfa drugs) which competitively inhibit bacterial synthesis of THF.
Amino acid biosynthesis and related products
Amino acids are not only the components of proteins, but also precursors to various compounds
including neurotransmitters, hormones and porphyrins.
Essential and nonessential amino acids in humans

Essential amino acids --- Amino acids that are not synthesized in human bodies.
- Plants and microorganisms can make essential amino acids.

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Takusagawas Note

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Chapter 20: Amino acid metabolism

Nonessential amino acids --- Amino acids that are synthesized in human bodies.
- These amino acids are synthesized from intermediates of glycolysis and the citric acid cycle.
Glucose-6-phosphate
Fructose-6-phosphate

Triose-3-phosphate
Glycerate-3-phosphate

Serine

Glycine

Asparagine Phosphoenolpyruvate
Aspartate

Pyruvate

Cystine

Alanine

Oxaloacetate

C.A.C. -Ketoglutarate

Glutamate

Glutamine
Proline

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Chapter 20: Amino acid metabolism

17

Takusagawas Note

Details of syntheses of Ala, Asp, Glu, Asn, and Gln are shown in Fig. 24-41.

Donor amino group

Glutamine synthetase is a central control point in nitrogen metabolism, since glutamine is the
amino group donor in the formation of many biosynthetic products as well as being a storage
form of ammonia.
- is 12 subunits protein (bacteria).
- is inhibited by two mechanisms:
1. Feedback inhibition. (In general, the final product inhibits the first reaction)
- His, Try, carbamoyl phosphate, AMP, CTP, glucosamine-6-phosphate which are all end
products of pathways leading from glutamine (i.e., receive amide nitrogen from glutamine)
are allosteric inhibitors.
- Ala, Ser, Gly inhibit by reflecting the cells high nitrogen level, i.e., Ala, Ser and Gly are
synthesized only the citric acid cycle is saturated.
- When the citric acid cycle is saturated, biosyntheses are started.

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Chapter 20: Amino acid metabolism

18

Takusagawas Note

2. Covalent modification.
- Adenylylation - deadenylylation and uridylylation - deuridylylation
Under conditions of nitrogen excess:
1. High [glutamine] activates uridylyl-removing enzyme.
2. Uridylyl-removing enzyme catalyzes deuridylylation of adenylyltransferase (PII-4UMP
PII).
3. Under a high [glutamine/-ketoglutarate] ratio, the PII catalyzes adenylylation of glutamine
synthetase, and inactivates it.
Under conditions of nitrogen limitation:
1. High [-Ketoglutarate] activates uridylyltransferase.
2. Uridylyltransferase catalyzes uridylylation of adenylyltransferase (PII PII-4UMP).
3. The uridylylated adenylyltransferase (PII-4UMP) catalyzes deadenylylation of glutamine
synthetase, and activates it.
4. Activated glutamine synthetase catalyzes glutamate to glutamine reaction.

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Chapter 20: Amino acid metabolism

19

Takusagawas Note

A specific tyrosine residue of adenylyltransferase (PII) is:

- uridylylated by uridylyltransferase (PII-4UMP is an active for deadenylylation).
- deuridylylated by uridylyl-removing enzyme (PII is an active for adenylylation).
Similarly, a specific tyrosine residue of glutamine synthetase is:
- adenylylated by deuridylylated adenylyltransferase (PII). Adenylylated enzyme is inactive.
- deadenylated by uridylylated adenylyltransferase (PII-4UMP). Deadenylylated enzyme is
active.

Glutamine

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Chapter 20: Amino acid metabolism

20

Glutamate is the precursor of proline, ornithine, and arginine

20

Takusagawas Note

Chapter 20: Amino acid metabolism

21

Takusagawas Note

Serine, cysteine, and glycine are derived from 3-phosphoglycerate

Gly

Cys

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Chapter 20: Amino acid metabolism

22

Takusagawas Note

S-adenosylmethionine (SAM) is synthesized from methionine and ATP

- SAM is the major methyl group donor molecule, and by releasing the methyl group, SAM
becomes S-adenosylhomocysteine.
Donor methyl
group

High level of homocysteine is one of the risk factors for coronary heart disease (heart
attack).

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Chapter 20: Amino acid metabolism

Takusagawas Note

Glycine is synthesized from serine by removing CH2OH group.

L-Serine

Serine hydroxymethyl
transferase

Glycine

5,10-methylene THF

THF

Tyrosine is synthesized from phenylalanine

L-Phe

Phenylalanine-4monooxygenase

O2 + tetrahydrobiopterin

dihydrobiopterin

NADP+

L-Tyr

NADPH + H+

Genetic disease, phenylketonuria is caused by less active or inactive phenylalanine-4monooxygenase. This disease produces abnormal level of phenylpyruvate in urine, since
phenylalanine is converted to phenylpyruvate instead of L-tyrosine.
Phe

Tyr

Phenylpyruvate
Amino acids are precursors of porphyrins, amines and peptides (glutathione)
Porphyrin synthesis
Porphyrins are derived from succinyl-CoA and glycine
Gly + Succinyl-CoA -Aminolevulinate (ALA) + CO2 + CoASH
- PLP is involved in the catalytic reaction.
-

Pyrrole ring is the product of two ALA molecules.

2ALA Porphobilinogen (PBG)

Uroporphyrinogen III is synthesized from four PBGs.

4PBG Hydroxymethylbilane Uroporphyrinogen III

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Chapter 20: Amino acid metabolism

24

Takusagawas Note

Overall heme biosynthesis is taken place in both mitochondrion and cytosol.

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Chapter 20: Amino acid metabolism

25

Takusagawas Note

There are several genetic defects in heme biosynthesis:

1. Uroporphyrinogen III cosynthase deficiency = congenital erythropoietic porphyria
Red urine, reddish teeth, photosensitive skin, increased hair growth.
2. Ferrochelatase deficiency = erythropoietic porphyria
Amine synthesis (Mainly decarboxylation by PLP dependent enzymes)
Some of amines are important neurotransmitters and hormones.
Biosynthesis of -aminobutyric acid (GABA, neurotransmitter), histamine (allergic response),
and serotonin (neurotransmitter)are shown below.

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Chapter 20: Amino acid metabolism

26

Epinephrine, norepinephrine and dopamine biosyntheses

HO
X
HO

C CH2

NH R

H
X = OH, R = CH3 Epinephrine
X = OH, R = H
Norepinephrine
X = H, R = H
Dopamine

Tyrosine is the precursor of these hormones.

Parkinsons disease

26

Takusagawas Note

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Chapter 20: Amino acid metabolism

-

Takusagawas Note

Deficiency in dopamine production is associated with Parkinsons disease (deficiency of

tyrosine hydroxylase). L-DOPA has been used to treat Parkinsons disease.
In melanocytes:
-

COO
tyrosinase

Tyr
O2

H2O

CH2 CH

tyrosinase

DOPA

NH3
O2

H2O

O
O
phenyl-3,4-quinone

polymerization
Melanine (black skin pigment)
Tyrosine hydroxylase (tyrosinase) is an important enzyme.

Glutathione

Important functions of GSH is elimination H2O2 and reduction of protein thiol-disulfied.

thiol transferase
S
SH
Protein
Protein
S
SH
2GSH
GSSG

27