Amino Acid Metabolism

•Metabolism of the 20 common amino acids is

considered from the origins and fates of their:
(1) Nitrogen atoms
(2) Carbon skeletons
•For mammals:
Essential amino acids must be obtained from diet
Nonessential amino acids - can be synthesized
 Non-Essential Amino Acids in
Humans
• Not required in diet
• Can be formed from α-keto acids by
transamination and subsequent reactions
• Alanine • Glycine
• Asparagine • Proline
• Aspartate • Serine
• Glutamate • Cysteine (from Met*)
• Glutamine • Tyrosine (from Phe*)
* Essential amino acids
 Essential Amino Acids in
Humans
• Required in diet
• Humans incapable of forming requisite
carbon skeleton
• Arginine* • Lysine
• Histidine* • Methionine
• Isoleucine • Threonine
• Leucine • Phenylalanine
• Valine • Tryptophan
* Essential in children, not in adults
. Fournisseur de peu d’énergie (10%)

. AA se jettent dans le cycle de Krebbs

. AA pour synthèse de protéines

. Catabolisme des aa absent chez les plantes

Catabolisme
• Whole proteins are not absorbed
– Too large to pass through cell membranes
intact
– Some combined with carbohydrates
(glycoproteins) +
H O
H3N C C
• Digestive enzymes R H O
N C C
– Break peptide bonds H
R H O
N C C
• Secreted as inactive pre-enzymes H
R
O–

– Prevents self-digestion
• Initiated in stomach
– HCl from parietal cells
• Stomach pH 1.6 to 3.2
• Denatures 40, 30, and 20 structures
– Pepsinogen from chief cells

• Cleaves at phenylalanine, tyrosine, tryptophan

Aromatic amino acids

• Protein leaves stomach as mix of insoluble protein, soluble

protein, peptides and amino acids
 Protein Digestion – Small Intestine

• Pancreatic enzymes secreted

– Trypsinogen
– Chymotrypsinogen
– Procarboxypeptidase
– Proelastase
– Collagenase
 – Small Intestine

• Zymogens must be converted to active form

– Trypsinogen Trypsin
• Endopeptidase
– Cleaves on carbonyl side of Lys & Arg
– Chymotrypsinogen Chymotrypsin
• Endopeptidase
– Cleaves carboxy terminal Phe, Tyr and Trp
– Procarboxypeptidase Carboxypeptidase
• Exopeptidase
– Removes carboxy terminal residues
Endo dans
Exo à partir des extrémités
Rate of an enzymatic reaction as a function
of temperature and pH
Zymogen activation (Lehninger 8-31)
 Specificity pocket explains protease
specificity (residues 189, 216 and 226)

Chymotrypsin Trypsin Elastase

aromatic basic small, uncharged

Diagram from Branden & Tooze, 1991

 Protein Digestion
• Small intestine (brush border)
– Aminopeptidases
• Cleave at N-terminal AA

– Dipeptidases
• Cleave dipeptides

– (Enterokinase or enteropeptidase)
• Trypsinogen → trypsin
• Trypsin then activates all the other enzymes
 Peptide Absorption

• Form in which the majority of

protein is absorbed
• More rapid than absorption
of free amino acids
• Coupled to H gradient
– Na/H exchange maintains
gradient
• Metabolized into free amino
acids in enterocyte
• Only free amino acids
absorbed into blood
• AA exogène >>>>>>>>>foie
• AA endogène

• Ammoniaque:
Toxique
Rare organisme qui transforment N2 en
ammonique
 The Nitrogen Cycle and Nitrogen
Fixation

• Nitrogen is needed for amino acids, nucleotides

• Atmospheric N2 is the ultimate source of
biological nitrogen
• Nitrogen fixation: a few bacteria possess
nitrogenase which can reduce N2 to ammonia
• Nitrogen is recycled in nature through the
nitrogen cycle
The Nitrogen cycle
 Nitrogenase

• An enzyme present in Rhizobium bacteria that

live in root nodules of leguminous plants
• Some free-living soil and aquatic bacteria also
possess nitrogenase
• Nitrogenase reaction:
N2 + 8 H+ + 8 e- + 16 ATP
2 NH3 + H2 + 16 ADP + 16 Pi
 Assimilation of Ammonia

• Ammonia generated from N2 is assimilated into

low molecular weight metabolites such as
glutamate or glutamine
• At pH 7 ammonium ion predominates (NH4+)
 Ammonia Is Incorporated into
Glutamate
• Reductive amination of α-ketoglutarate by
glutamate dehydrogenase occurs in plants,
animals and microorganisms
• In mammals & plants, located in mitochondria.
 Glutamine Is a Nitrogen
Carrier in Many Biosynthetic
Reactions
• A second important route in assimilation of
ammonia is via glutamine synthetase
Glutamate synthase transfers
a nitrogen to α-ketoglutarate
Prokaryotes & plants
Alternate amino acid production
in prokaryotes
 How some enzymes transfer
ammonia from glutamine
• CP synthetase has 3 active sites connected by a
tunnel running through the interior
• Protects intermediates from being degraded by
water
 Carbamoyl phosphate
synthase backbone structure
• Tunnel connecting active sites (blue wire)
 C. Regulation of Glutamine
Synthetase in E. coli

• Glutamine synthetase (GS) plays a critical role

in nitrogen metabolism
• E. coli enzyme regulated by:
(1) Cumulative feedback inhibition
(9 allosteric inhibitors with additive effects)
(2) Covalent modification
(3) Regulation of enzyme synthesis
Allosteric inhibition of GS in E.
coli
Regulation of E. coli GS by
covalent modification
 Regulation of mammalian GS

• Regulation not as extensive as in microorganisms

• No covalent regulation
• Allosteric inhibitors: glycine, serine, alanine, and
carbamoyl phosphate
• Allosteric activator: α-ketoglutarate
 Transamination Reactions

• Transfer of an amino group from an α-amino

acid to an α-keto acid
• In amino acid biosynthesis, the amino group of
glutamate is transferred to various α-keto acids
generating α-amino acids
• In amino acid catabolism, transamination
reactions generate glutamate or aspartate
Transamination reactions
 H
- -
R1 C COO + R2 C COO
+
NH3 O
Transaminases
Transaminase
(aminotransferase
s) catalyze the H
reversible reaction R1 C COO
-
+ R2 C COO
-

at right. +
O NH3

There are multiple transaminase enzymes which

vary in substrate specificity.
Some show preference for particular amino acids
or classes of amino acids as amino group donors,
and/or for particular α-keto acid acceptors.
Dans le foie COO− COO−

COO− CH2 COO− CH2

CH2 CH2 CH2 CH2

HC NH3+ + C O C O + HC NH3+

COO− COO− COO− COO−

aspartate α-ketoglutarate oxaloacetate glutamate

Aminotransferase (Transaminase)

Example of a Transaminase reaction:

Aspartate donates its amino group,
becoming the α-keto acid oxaloacetate.
α-Ketoglutarate accepts the amino group,
becoming the amino acid glutamate.
Transaminases equilibrate amino groups
among available α-keto acids.
This permits synthesis of non-essential amino
acids, using amino groups from other amino
acids & carbon skeletons synthesized in a cell.
Thus a balance of different amino acids is
maintained, as proteins of varied amino acid
contents are synthesized.
Although the amino N of one amino acid can be
used to synthesize another amino acid, N must
be obtained in the diet as amino acids
(proteins).
 H O
O− C
−O H2
P C OH
O
O
+
N CH3
H
pyridoxal phosphate (PLP)

The prosthetic group of Transaminase is

pyridoxal phosphate (PLP), a derivative
of vitamin B6.
 H
R C COO− Enz

NH2 (CH2)4
Amino acid N+
O− HC H
−O H2
P C O−
O
O
+
N CH3
H
Enzyme (Lys)-PLP Schiff base

In the resting state, the aldehyde group of

pyridoxal phosphate is in a Schiff base linkage
to the ε-amino group of an enzyme lysine side-
chain.
 Enz−Lys−NH2 H
R C COO−

N+
O− HC H
−O H2
The α-amino P C O−
group of a O
O
substrate amino
acid displaces the +
N CH3
enzyme lysine, to H
form a Schiff base Amino acid-PLP Shiff base (aldimine)
linkage to PLP.
The active site lysine extracts H+, promoting
tautomerization, followed by reprotonation &
hydrolysis.
 O
Enz−Lys−NH2
NH2 R C COO−
α-keto acid
O− CH2
−O H2
P C OH
What was O
O
an amino +
acid leaves N CH3
as an α- H
keto acid. Pyridoxamine phosphate (PMP)

The amino group remains on what is now

pyridoxamine phosphate (PMP).
A different α-keto acid reacts with PMP and the
process reverses, to complete the reaction.
 Enz−Lys−NH2 H
R C COO−

N+
O− HC H
−O H2
P C O−
O
O
+
N CH3
H
Amino acid-PLP Shiff base (aldimine)

Several other enzymes that catalyze metabolism

or synthesis of amino acids also utilize PLP as
prosthetic group, and have mechanisms involving a
Schiff base linkage of the amino group to PLP.
In addition to equilibrating amino groups among
available α-keto acids, transaminases funnel
amino groups from excess dietary amino acids to
those amino acids (e.g., glutamate) that can be
deaminated.
Carbon skeletons of deaminated amino acids can
be catabolized for energy, or used to synthesize
glucose or fatty acids for energy storage.
Only a few amino acids are deaminated directly.
 NH3+
H2 H2
−
OOC C C C COO−
Glutamate glutamate
H +
NAD(P)
Dehydrogenase H2O
catalyzes a major NAD(P)H
O
reaction that H2 H2
effects net −
OOC C C C COO− + NH4+
removal of N α-ketoglutarate
from the amino Glutamate Dehydrogenase
acid pool.
It is one of the few enzymes that can use NAD+ or
NADP+ as e− acceptor.
Oxidation at the α-carbon is followed by hydrolysis,
releasing NH4+.
 +
Amino acid α-ketoglutarate NADH + NH4

+
α-keto acid glutamate NAD + H2O

Transaminase Glutamate
Dehydrogenase

Summarized above:
The role of transaminases in funneling amino N
to glutamate, which is deaminated via Glutamate
Dehydrogenase, producing NH4+.
 +
H2O H2O NH4 O
H
HO CH2 C COO− H2C C COO− H3C C COO−

NH3+ NH3+
serine aminoacrylate pyruvate
Serine Dehydratase
Some other pathways for deamination of amino
acids:
1. Serine Dehydratase catalyzes:
serine pyruvate + NH4+
 2. Peroxisomal L- and D-amino acid oxidases catalyze:
amino acid + FAD + H2O
a-keto acid + NH4+ + FADH2
FADH2 + O2 FAD + H2O2
Catalase catalyzes: 2 H2O2 2 H2O + O2

Postulated mechanisms for toxicity of high [ammonia]:

1. High [NH3] would drive Glutamine Synthase:
glutamate + ATP + NH3 glutamine + ADP + Pi
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)+ α-ketoglutarate +
NAD(P)H + NH4+
The resulting depletion of α-ketoglutarate, an essential Krebs Cycle intermediate, could
impair energy metabolism in the brain.
 O

H2N C NH2
urea

Most terrestrial land animals convert excess

nitrogen to urea, prior to excreting it.
Urea is less toxic than ammonia.
The Urea Cycle occurs mainly in liver.
The 2 nitrogen atoms of urea enter the Urea Cycle
as NH3 (produced mainly via Glutamate
Dehydrogenase) and as the amino N of aspartate.
The NH3 and HCO3− (carbonyl C) that will be part of
urea are incorporated first into carbamoyl
phosphate.
 Urea Cycle

• Overall reaction O
||
2 NH3 + CO2 → → H2N–C–NH2 + H2O

!"
 HCO3−
ATP
Carbamoyl Phosphate ADP
Synthase (Type I) catalyzes O
a 3-step reaction, with HO C OPO32−
carbonyl phosphate and NH3 carbonyl phosphate
carbamate intermediates. Pi
O
Ammonia is the N input.
H2N C O−
The reaction, which
ATP carbamate
involves cleavage of 2 ~P
ADP
bonds of ATP, is essentially
O
irreversible.
H2N C OPO32−
carbamoyl phosphate
 O O C NH2
Urea Cycle NH3+ H2N C OPO32− NH
Enzymes in CH2 carbamoyl CH2
mitochondria: phosphate
CH2 citrulline
Pi CH2
1. Ornithine
CH2 1
Trans- CH2
carbamylase HC NH3+ COO−
HC NH3+
Enzymes in Urea Cycle
COO− CH2
COO−
cytosol: ornithine ATP 2 HC NH2
2. Arginino- AMP + PPi
Succinate O 4 H2O COO−
−
COO
Synthase H2N C NH2 aspartate
3. Arginino- urea H2N NH2+ CH2
succinase C H
3 HC N C NH2+
4. Arginase. NH COO− NH
CH2
COO− CH2 arginino-
arginine CH2 succinate
HC CH2
CH2 CH2
CH
HC NH3+ NH3+
COO− HC
COO− fumarate COO−
 NH3 + + Asp NH3+
NH3 -CHCH2CO2 - +
-
NH2 CONH CH 2 CH 2 CH 2 CHCO 2 CO2 - H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 -
Citrulline
Arginosuccinate NH-CHCH2 CO2-
synthase
CO2- Arginosuccinate
Ornithine
Transcarbamoylase
(mitochondria) H CO2-

Fumarate
NH3+ Urea
+ -
O2 C H

H3 NCH 2 CH 2 CH 2 CHCO 2 - H2NCONH2 Argino-

succinase
Ornithine TCA Cycle
NH2 NH3 +
Arginase +
H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 -
Arginine
 cytosol
The complete Urea mitochondrial matrix
Cycle is significantly only
in liver.
carbamoyl phosphate
However some enzymes
of the pathway are in Pi
other cells and ornithine citrulline

ornithine citrulline
urea aspartate
arginine argininosuccinate
fumarate
tissues where they generate arginine & ornithine, which are precursors
for other important molecules.
E.g., Argininosuccinate Synthase, which catalyzes synthesis of the
precursor to arginine, is in most tissues.
Mitochondrial Arginase II, distinct from the cytosolic Urea Cycle Arginase,
cleaves arginine to yield ornithine.
 cytosol
mitochondrial matrix

carbamoyl phosphate
Pi
ornithine citrulline

ornithine citrulline
urea aspartate
arginine argininosuccinate
fumarate
For each cycle, citrulline must leave the mitochondria, and ornithine must
enter the mitochondrial matrix.
An ornithine/citrulline transporter in the inner mitochondrial membrane
facilitates transmembrane fluxes of citrulline & ornithine.
 NH2 NH2 NH2
+
C NH2 C N OH C O

NH NH NH
NADPH NADP+ 1/2 NADPH 1/2 NADP+
CH2 CH2 CH2 + NO
CH2 O2 H2O CH2 O2 H2O CH2

CH2 CH2 CH2

+
H3N CH COO− +
H3N CH COO−
+
H 3N CH COO−
arginine hydroxyarginine citrulline
Nitric Oxide Synthase
Arginine is a constituent of proteins, & is precursor for synthesis of creatine and
nitric oxide (·NO).
·NO is a short-lived signal molecule with diverse roles in different cell types:
regulating smooth muscle contraction, gene transcription, metabolism, &
neurotransmission.
Many effects of ·NO arise from its activation of a soluble cytosolic Guanylate Cyclase
that synthesizes cyclic-GMP.
Au niveau des muscles

Glutamate a-Ketoglutarate
+ +
Pyruvate Alanine

NH4+ foie

Glutamate a-Ketoglutarate
+ +
Pyruvate Alanine

Gluconéogénese
 +
Incorporation of Into NH4
Organic Compounds
+ - Carbamoyl
1) NH4 + HCO3 + 2 ATP Phosphate NH2CO2PO3-2 + 2 ADP +
Synthase I +
Carbamoyl Phosphate Pi + 2 H
(CPS-I)

TCA Cycle

O Glutamate
+ NH3+
2) NH4 + -
O 2 CCH 2 CH 2 CCO 2 -
dehydrogenase
-
O 2 CCH 2 CH 2 CHCO 2 -
α-Ketoglutarate NADPH +
H+
NADP +
Glutamate
 +
Incorporation of Into NH4
Organic Compounds

3) NH3+ +
-
O 2 CCH 2 CH 2 CHCO 2 - + NH4 + 2 ATP
Glutamate Glutamine
Synthase
Mg++

O NH3 +
H2 NCCH 2 CH 2 CHCO 2 -

Glutamine
N of glutamine donated to other compounds
in synthesis of purines, pyrimidines,
and other amino acids
 Biosynthesis of Amino Acids:
Transaminations
α-Keto Acid2
Amino Acid1 +α α-Keto Acid1
Amino Acid2 +α

NH3+ O
-
O 2 CCH 2 CH 2 CHCO 2 - + R-CCO 2 -
Glutamate
Pyridoxal phosphate (PLP)-
Dependent Aminotransferase

O
NH2
-
O 2 CCH 2 CH 2 CCO 2 - +
R-CHCO 2 -
α-Ketoglutarate
Transaminations: Role of PLP
CO2 -

CHO H N CHCH2 CH2 CO2-

C
HO CH2 OPO3-2
HO CH2 OPO3-2

H3 C N
+ H3 C N
H +
NH3 + H2 O H
- -
O 2 CCH 2 CH 2 CHCO 2 Tautomerization

O CO2 -
-
O 2 CCH 2 CH 2 CCO 2 - N CCH2 CH2 CO2-
CH2 NH2 CH2
HO CH2 OPO3-2 HO CH2 OPO3-2

H3 C N H2 O H3 C
+ N
+
H H
 Transaminations
Glutamate-Pyruvate
Aminotransferase
Glutamate (Alanine Transferase ALT) α-Ketoglutarate
+ +
Pyruvate Alanine
Glutamate-Oxaloacetate
Aminotransferase
(Aspartate Transferase AST)
Glutamate α-Ketoglutarate
+ +
Oxaloacetate Aspartate
 Ketogenic Amino Acids
• Metabolized to acetyl CoA or
acetoacetate
• Isoleucine • Lysine
• Leucine • Phenylalanine
• Threonine • Tyrosine
• Tryptophan
 Amino Acids Formed From α-
Ketoglutarate
O
-
O 2 CCH 2 CH 2 CCO 2 -
α-Keto- 4 Steps
Transamination or
glutarate Glutamate
dehydrogenase
+ CO2 -
N
NH3+
- - 5 Steps H H Proline
O 2 CCH 2 CH 2 CHCO 2
Glutamate NH3 +
+
Glutamine
H3 NCH 2 CH 2 CH 2 CHCO 2 - Ornithine
synthase
Urea Cycle
O NH3 + NH2 NH3 +
+
H2 NCCH 2 CH 2 CHCO 2 - H2 N=C-HNCH 2 CH 2 CH 2 CHCO 2 - Arginine
Glutamine Guanidino group
 Use of Keto Acids for Energy
• Keto acids can
– Enter the TCA cycle and be broken down to
CO2 and H2O with release of energy
– Be used for gluconeogenesis
• Some, not all amino acids
• In liver (and kidney)
– Lipogenesis
– Ketogenesis
• Ketone bodies (acetoacetate, acetyl-CoA)
• Used as energy source in various tissues
 Disposal of NH3
• NH3 is very toxic and must be
detoxified and excreted from the body
– Fish: elimination au niveau des branchies NH3
Organismes ammnoteliques
– Mammals: Urea plus soluble et moins toxique que NH3
Organismes ureoteliques
– Birds: Uric acid
Organismes ureotiliques
• Synthesis of uric acid
– Same pathway as for purines
• Synthesis of urea—the urea cycle
– Detoxifies NH3 to urea
– Synthesizes arginine
Boisen et al. (2000)
 Photosynthesis
Light energy is captured and stored as chemical
potential energy in the covalent bonds of
carbohydrate molecules.
The overall chemical reaction for photosynthesis is:
6 CO2 + 6 H2O + light energy → C6H12O6 + 6 O2

Photosynthesis occurs in plants, algae, and certain

types of bacteria, all of which are autotrophs
(literally, “self-feeders”)
 Chloroplasts are the
sites of photosynthesis
in plants.
Evidence that
chloroplasts split
water molecules
enabled researchers to
track atoms through
photosynthesis.
The Pathways of Photosynthesis
 The oxygen
released by plants
during
photosynthesis
comes from
WATER, not from
carbon dioxide.
An overview of photosynthesis: cooperation of the
light reactions and the Calvin cycle.
Structures
 Visible light is only
a small portion of
the electromagnetic
spectrum. Visible
light ranges from
about 390 nm to
about 760 nm.

To our eyes, 400 nm light appears violet, 500 nm is blue-

green, and 600 nm is orange-red. Remember, what we see
is light REFLECTED off of objects.
Because the Earth’s
atmosphere absorbs
most infrared and
ultraviolet light,
most of the light
that reaches the
Earth’s surface is
visible light. This
visible light is then
used in
photosynthesis to
build complex
molecules.
 Mechanisms of
Photosynthesis
Substances needed:

Photons … sunlight
CO2 … atmosphere
H2O … soil, rain
Chlorophyll … chloroplasts
Light-dependent reactions occur in two steps:
1. Conversion of solar energy (photons) to chemical energy
(high energy ATP) phosphorylation
2. ATP is used to join CO2 and H2O → PGAL → Glucose
 Light-dependent reactions
• Are carried out by molecules in the thylakoid
membranes.
• Convert light energy to the chemical energy of ATP
and NADPH
• Split H2O and release O2 atmosphere.
• Involves two PHOTOSYSTEMS:
• Photosystem I absorbs light at 700 nm and is called
P700 (P stands for “pigment”.)
• Photosystem II absorbs light at 680nm and is called
P680
 pQ plastoquinone PQH2
Pc plastocyanine
Fd ferrédoxine

Phéophytine

2
Light-dependent reactions,
(photophosphorylation)
- generates ATP and
NADPH
- occurs in the thylakoid
membranes of the
chloroplast
Light-independent
(Calvin cycle)
- uses ATP and electrons
from light reactions along
with CO2
- occurs outside the
thylakoid membrane.
 H 2C OPO32− H 2C OPO32− H2C OPO32−
1
−
O C2 O C CO2 HO C CO2−
O O−
H C OH C OH C O C
3

H C OH H+ H C OH H C OH H2O H C OH
4

H 2C OPO32− H 2C OPO32− H2C OPO32− H 2C OPO32−

5
ribulose-1,5- enediolate β-keto 3-phosphoglycerate
bisphosphate intermediate intermediate (2)

RuBP Carboxylase - postulated mechanism:

Extraction of H+ from C3 of ribulose-1,5-bisphosphate promotes formation of
an enediolate intermediate.
Nucleophilic attack on CO2 leads to formation of a β-keto acid
intermediate, that reacts with water and cleaves to form 2 molecules of 3-
phosphoglycerate.