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Chapter 20: Amino acid metabolism
Takusagawa’s 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
O
-
α-KA1
O
R CH C O
L-amino acid
-
-
O
COO
R1 C COO + R2 CH NH2
CH NH2 + R2 C COO
AA1
α-KA2
H3N+
-
O
O
C CH2 CH2 C C O
α-Ketoglutarate
-
-
H3N
O
R C
C O
α-Keto acid
O
C
+
O
CH2 CH C O
Aspartate
-
transaminase
O
-
H3N
O
O
transaminase
O
AA2
-
O
C
+
O
CH2 CH2 CH C O
Glutamate
-
-
O
O
O
C
CH2 C C O
Oxaloacetate
O
-
Then Asp →→ Urea
Oxidative deamination
H3N+
O
R CH C O
L-amino acid
O
-
-
O
O
O
C CH2 CH2 C C O
α-ketoglutarate NH3
transaminase
O
O
R C
C O
α-keto acid
H3N+ O
O
-
O
C CH2 CH2 CH C O
glutamate
Urea
1
NADH + H+
glutamate
dehydrogenase
+
NAD + H2O
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Chapter 20: Amino acid metabolism
Takusagawa’s 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 ↔ α-Keto acid + Enzyme-NH2
2. α-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 (ε-amino group) of enzyme.
E-Lys + PLP ↔ E-PLP
(CH2)4
H
2-
O3PO
H2
C
C
N
H
-
O
+
N
H
2
CH3
Enzyme
Chapter 20: Amino acid metabolism
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Takusagawa’s Note©
Stage I: Conversion of an amino acid to α-keto Acid
1. Transimination: Amino acid’s 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 α-keto acid-PMP by the active-site Lys
catalyzed removal of the amino acid α-hydrogen and protonation of PLP atom C4’.
AA-PLP ↔ α-Keto acid-PMP
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Chapter 20: Amino acid metabolism
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Takusagawa’s Note©
3. Hydrolysis: α-Keto acid-PMP is hydrolyzed to PMP and α-Keto acid.
α-Keto acid-PMP + H2O ↔ α-Keto acid + PMP
Stage II: Conversion of an α-keto acid to an amino acid (reverse reactions of stage I)
3’. α-Keto acid + PMP ↔ α-Keto acid-PMP
2’. α-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 β- or γ-carbon
4. Racemization (D ↔ L)
5. Others
4
H
Cγ
2
Cβ Cα COO
-
HN
1
-
HC
OH
O3P O
N
H
4
NH2 E
CH3
Takusagawa’s Note©
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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
C
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.
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Chapter 20: Amino acid metabolism
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Takusagawa’s 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
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Chapter 20: Amino acid metabolism
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Takusagawa’s 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 cycle’s substrate (Glu) and product (malate) produce 2 NADH (= 6
ATP) as shown in Fig. 24-7.
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Chapter 20: Amino acid metabolism
Takusagawa’s 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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9
Takusagawa’s 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 acid’s NH3 in muscle is transferred to pyruvate.
- The aminated pyruvate, Ala, is transported to liver where the NH2 is transported to αketoglutarate.
- The aminated α-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.
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Chapter 20: Amino acid metabolism
Takusagawa’s Note©
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Amino acid’s 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.
α-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
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Takusagawa’s 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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Takusagawa’s 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
Takusagawa’s 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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Takusagawa’s Note©
Reactions involved in THF are oxidation-reduction, cyclization and hydrolysis
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Takusagawa’s 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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Takusagawa’s 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. α-Ketoglutarate
Glutamate
Glutamine
Proline
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Chapter 20: Amino acid metabolism
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Takusagawa’s 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 cell’s 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
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Takusagawa’s 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/α-ketoglutarate] ratio, the PII catalyzes adenylylation of glutamine
synthetase, and inactivates it.
Under conditions of nitrogen limitation:
1. High [α-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
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Takusagawa’s 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
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Glutamate is the precursor of proline, ornithine, and arginine
20
Takusagawa’s Note©
Chapter 20: Amino acid metabolism
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Takusagawa’s Note©
Serine, cysteine, and glycine are derived from 3-phosphoglycerate
Gly
Cys
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Chapter 20: Amino acid metabolism
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Takusagawa’s 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
Takusagawa’s 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 ⎯⎯→ δ-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
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Takusagawa’s Note©
Overall heme biosynthesis is taken place in both mitochondrion and cytosol.
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Chapter 20: Amino acid metabolism
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Takusagawa’s 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 γ-aminobutyric acid (GABA, neurotransmitter), histamine (allergic response),
and serotonin (neurotransmitter)are shown below.
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Chapter 20: Amino acid metabolism
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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.
Parkinson’s disease
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Takusagawa’s Note©
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Chapter 20: Amino acid metabolism
-
Takusagawa’s Note©
Deficiency in dopamine production is associated with Parkinson’s disease (deficiency of
tyrosine hydroxylase). L-DOPA has been used to treat Parkinson’s 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
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