Biochemistry I
Lecture 6 2008 (J.S.)
Amino acid metabolism I
Catabolism of proteins
General catabolism of amino acids
2
Digestion of dietary proteins in the GIT
Protein digestion begins in the stomach, where the acidic environment
favours protein denaturation. Proteins undergo the primary proteolysis
catalyse by gastric pepsin (the optimal pH 2).
Degradation continues in the lumen of the intestine owing to the activity
of pancreatic proteolytic enzymes secreted as inactive proenzymes
and converted into active enzymes within the intestinal lumen..
Digestion is then completed by aminopeptidases located in the plasma
membrane (brush border) of the enterocytes.
Single amino acids, as well as di- and tripeptides, are transported
into the intestinal cells from the lumen and released into the blood.
3
The generation of trypsin leads to the activation of other proenzymes:
Enteropeptidase secreted by the mucosa of duodenum initiates the activation
of the pancreatic proenzymes by activating trypsin, which then activates other
proenzymes.
4
Proteolytic activation of chymotrypsinogen
The active site for binding a part of the substrate is not fully formed in the
chymotrypsinogen. Proteolysis enables the formation of the substrate-binding site.
5
Proteolytic enzymes of the GIT
attack peptide bonds of a number of amino acids, but they exhibit the
preference for particular types of peptide bonds:
Proteinases (formerly called endopeptidases): Preferentially attacks the bond after:
Pepsin aromatic (Phe, Tyr) and acidic amino acids (Glu, Asp)
Trypsin basic amino acids (Arg, Lys)
Chymotrypsin hydrophobic (Phe, Tyr, Trp, Leu) and acidic AA (Glu, Asp)
Elastase an amino acid with a small side chain (Gly, Ala, Ser)
Peptidases (formerly exopeptidases):
Carboxypeptidase A nearly all amino acids (not Arg and Lys)
Carboxypeptidase B basic amino acids (Arg, Lys)
Leucine aminopeptidase nearly all amino acids
Prolidase proline
Dipeptidase splits only dipeptides
6
The absorption of amino acids in the gut
The absorption of amino acids and peptides is an active proces,
obligatorily coupled to the uptake of Na+
. It is driven by the large
difference in Na+
concentration across the brush border membrane, that
is maintained by the Na+
,K+
-ATPase.
There are at least seven different Na+
-linked amino acid transporters of
different but overlapping specificities, and also separate transporters for
di- and tripeptides.
Na+
7
Degradation of intracellular proteins
– eliminates abnormal proteins,
– permits the regulation of cellular metabolism by elimination of some
enzymes and/or regulatory proteins that represent important
metabolic control points.
Normal intracellular proteins
are eliminated at rates that depend on
their identity.
Long-lived proteins, under
physiological conditions, are degraded
at nearly constant rates, mostly
nonselectively; nutritional deprivation
increases rates of degradation and it is
selective.
Short-lived proteins degradation is
provided selectively by cytosolic
ubiquitin systém, or by other systems
not yet known.
8
Systems which select proteins for degradation:
– The N-end amino acyl residue – in both prokaryotes and eukaryots the half-life
of a cytoplasmic protein varies with the identity of its N-terminal amino acyl:
– Some sequences of amino acyl residues in the primary structure.
Proteins rich in segments Pro-Glu-Ser-Thr (symbol PEST), so-called PEST proteins,
are rapidly degraded in the cytosol, t½ about 30 minutes.
During a prolonged fasting, proteins with sequences Lys-Phe-Glu-Arg-Gln (symbol KFERQ),
KFERQ proteins, in the tissues that atrophy in response to starvation (e.g. liver, kidney)
are specifically bound with the protein prp73, delivered to the lysosome, and degraded.
Cyclin destruction boxes – amino acid sequences that mark cell-cycle proteins for
destruction.
– A highly destabilizing N-terminal residue such as Arg and Leu favours rapid
ubiquitinization, the ubiquitin-tagged proteins are degraded in the proteasomes.
– Other, more complex systems?
9
1 Protein degradation in lysosomes
Lysosomes contain a large number of proteolytic enzymes, cathepsins, which
are active at pH 5 maintained by the vesicular H+
pump (and largely inactive at
cytosolic pH values).
Extracellular proteins that enter the cell via endocytosis are hydrolysed in the
phagolysosomes, which are formed by fusing the invaginated vesicles (simple
endosomes or coated vesicles covered with clathrin in receptor-mediated
endocytosis) with lysosomes.
Normal long-lived intracellular proteins are also degraded nonselectively in
the lysosomes (autophagy – the digestion of cytoplasmic proteins by the cell´s
own lysosomes).
The protein degradation in lysosomes is ATP-independent. It is probably of
less importance than the turnover of specific proteins in the ubiquitin pathway.
)
10
2 Cytosolic ubiquitin system (ATP-dependent)
C-end
The C-terminal glycine residue of ubiquitin (Ub)
becomes covalently attached to the ε-amino
groups of several lysine residues (isopeptide
bonds) on a protein destined to be degraded.
The energy for the formation of these isopeptide
bonds comes from ATP hydrolysis.
Three enzymes participate in the attachment of
ubiquitin to each protein:
Ubiquitin, a small protein (Mr 8500) present in all eukaryotic cells,
is the tag that marks proteins for destruction.
Ubiquitin is the most highly conserved protein known in
eukaryotes. It is identical in humans, toad, trout,
Drosophila. Yeast and human ubiquitin differs at only 3
of 76 amino acyl residues.
11
E 2
E 1
E 2
SH
SH
E 1
SH
E 1
AMP
ATP
E 1
PPi
E 3
Protein
E 2
ProteinH3N–
+
E 3
E 3
E 2
Ubiquitinated protein
Enzyme 1 – ubiquitin-activating
Enzyme 2 – ubiquitin-conjugating
Enzyme 3 – ubiquitin-protein ligase
The ubiquitination reaction is processive – chains of Ub are generated by the linkage of the
ε-amino group of lysine of one ubiquitin molecule to the terminal carboxylate of another.
Chains of four or more ubiquitins are particularly effective in signaling degradation.
The ubiquitin-tagged protein heads towards proteasome.
12
The proteasome
digests the ubiquitin-tagged proteins. This ATP-driven multisubunit
process spares ubiquitin, which is then recycled.
The 26S proteasome is a complex of a 20S proteasome (it exhibits
at least 5 different proteolytic activities), and two 19S regulatory
complexes.
26S proteasome
20S proteasome
19S complex
19S complex
20S proteasome
13
The 20S proteasome complex consists of 28 subunits arranged in four rings
of subunits that stack to form a structure resembling a barrel. The active
sites of the proteinases are on the interior of the barrel.
Substrates are degraded until they are reduced to peptides in lengths from
seven to nine amino acyl residues.
The peptide products are further degraded by other cytoplasmic proteases to
yield individual amino acids.
The 20S proteasome is a sealed barrel. Access to its interior is controlled by
two 19S regulatory complexes (each made up of 20 subunits), which bind
specifically to polyubiquitin chains and exhibit ATPase activity. ATP
hydrolysis may assist the 19S complex to unfold the substrate so that it can
be passed into the center of the proteasome.
The 19S complex also cleaves off intact ubiquitin molecules.
14
The dynamics of amino acid metabolism
and the protein turnover
DIETARY PROTEINS
approx. 80 g daily
THE INTESTINE
10 g / d loss in stool
AMINO ACID POOL
Free AA in plasma and cells
approx. 70g
PROTEOSYNTHESIS
260 g (up to 400 g) daily
Plasma proteins 20 g /d
Visceral proteins 50 g / d
Muscle proteins 50 g / d
70 g / d
Intestinal secretion
AMINO ACID
CATABOLISM
Protein breakdown
Specific products
140 g / d
Absorption
UREA
Urinary excretion
equivalent to 70 g protein / d
CO2 + H2O
15
The minimum protein requirement of adults
is above 0.4 g / kg daily, i.e. 28 g / d for a 70 kg man, supposing that the proteins
given will be utilized with maximal efficiency.
The recommended daily protein supply for adults
is about 0.8 g / kg, i.e. 56 g / d for a 70 kg man.
The protein requirement of children is larger than that of adults – 1.2-1.5 g / kg.
The effectiveness of different proteins in the diet
An empirical measure of the efficiency of a protein is the biological value –
the mass of body proteins that are synthesized from 100 g of a protein
(expressed as percentage).
It depends primarily on the sufficient content of the essential amino acids
which the body cannot make itself, and on the digestibility of a protein.
Essential amino acids
Valine Methionine
Leucine Phenylalanine
Isoleucine Tryptophan
Threonine Histidine (for children)
Lysine Arginine (for children)
16
Eggs 97
Milk 90
Beef 77
Pork 71
Casein 68
Oat flakes 62
Wheat 53 (low in lysine)
Beans 46 (low in methionine)
Lentils 45 (low in methionine)
Gelatine 25 (very low in tryptophan)
Examples of the biological values of proteins in some foods(%) :
Examples of protein content of foods (%) :
Cheese 25-30
Curd 30
Cottage cheese 20
Meat 20
Eggs 13
Cow´s milk 3.3
Legumes 25-30
Yeast 11
Pasta products 8-10
Rice 7
Potatoes 2
Fruit, vegetables 0.5-2
17
The amino acid degradation
The first step in degradation of many standard amino acids is the
removal of the α-amino group, i.e. deamination or transamination.
The product is mostly the corresponding 2-oxoacid (α-ketoacid) and
α-amino group is released as ammonia or ammonium ion.
Direct deamination of amino acids
Only few amino acids are deaminated directly:
Glutamate – oxidative deamination catalysed by glutamate dehydrogenase
Serin and threonine – deamination preceded by dehydratation
catalysed by serine dehydratase or threonine dehydratase.
Histidine – deamination by elimination of NH3 catalysed by histidine ammonialyase.
In peroxisomes, aerobic deamination of various amino acids
catalysed by amino acid oxidase is not very efficient.
Direct deamination is therefore unimportant for most L-amino acids; instead,
their amino groups are removed indirectly, by transamination.
18
Direct oxidative deamination of glutamate by dehydrogenation
Glutamate
NAD(P)+
NAD(P)H + H+
NH2
HOOC-CH-CH2-CH2-COOH
2-Iminoglutarate
2-Oxoglutarate
(α-Ketoglutarate)
NH
HOOC–C–CH2-CH2-COOH
HOOC–C–CH2-CH2-COOH
O
H2O
NH3
The reaction is catalysed by the mitochondrial enzyme
glutamate dehydrogenase (GLD). It requires either NAD+
or NADP+
as coenzyme,
and its activity in mitochondria is high.
The equilibrium favours glutamate
synthesis, but it is pulled in the direction
of deamination by the continuous
removal of NH3/NH4
+
.
19
Direct deamination of histidine by elimination (desaturation)
N
N
H
–CH2–CH–COOH
NH2
NH3
N
N
H
–CH=CH–COOH
Histidase
(L-Histidine ammonialyase)
Histidine Urocanic acid
(Urocanate)
Direct oxidative (aerobic) deamination of amino acids in peroxisomes
is catalysed by L-amino acid oxidase. In spite of this reaction seems to be similar to that catalysed
by glutamate dehydrogenase, the amino acid oxidases are flavoproteins which require O2 for reoxidation
of FADH2, and they produce hydrogen peroxide.
Catalase
H2O
NH3FAD FADH2
NH
R–C–COOH
NH2
R–CH-COOH R–C–COOH
O
O2
H2O + ½O2 H2O2
Amino acid Imino acid 2-oxoacid
(α-Ketoacid)
20
Transamination
Pyridoxal phosphate (PLP)
An aminotransferase reaction is shown on the next picture.
Transaminations are reactions with a "ping-pong“ mechanism:
the first product leaves the enzyme before the second substrate binds, in the meantime
pyridoxal phosphate binds the transferred amino group (as pyridoxamine phosphate).
The aldehyde group enables binding to α-amino group of an
amino acid so that aldimine intermediates (Schiff´s bases)
are formed.
The α-amino group of many amino acids that cannot be deaminated
directly is transferred to 2-oxoglutarate (α-ketoglutarate) to form
glutamate. The product glutamate is oxidatively deaminated by
glutamate dehydrogenase to yield ammonium ion NH4
+
.
These transaminations are reversible and can be used to synthesize
amino acids from 2-oxoacids and glutamate.
Transaminations are catalysed by aminotransferases, the prosthetic
group is pyridoxal phosphate.
21
H2– NH2
Pyridoxamine-P
The 1st
transfer
C
Amino acid
CH
NH2
COOHR
1st substrate
Oxoacid
R C–
O
COOH
1st product
Ketimine of AA
Aldimine of PLP
HC=O
Pyridoxal-P
–H2O
+H2O
-H2O
HC=O
Pyridoxal-P
The 2nd
transfer
HOOC–C–CH2-CH2-COOH
O
2-Oxoglutarate
2nd substrate
Glutamate
NH2
HOOC-CH-CH2-CH2-COOH
2nd product
Ketimine of αKG
Aldimine of PLP
+H2O
22
The product of transamination glutamate is then deaminated
directly by the action of glutamate dehydrogenase (GLD):
Transamination: Oxidative deamination of glutamate
2-Oxoglutarate
Among other aminotransferases, the two are important (e.g. assays in clinical
biochemistry, see Practicals):
aspartate aminotransferase (AST, L-aspartate:2-oxoglutarate aminotransferase),
which catalyses the reaction
aspartate + 2-oxoglutarate oxaloacetate + glutamate , and
alanine aminotransferase (ALT, L-alanine:2-oxoglutarate aminotransferase), which in
the liver catalyses the reaction
alanine + 2-oxoglutarate pyruvate + glutamate.
23
Indirect deamination of glutamate – so-called "purine nucleotide cycle"
In some tissues, in skeletal muscle particularly, glutamate is deaminated not directly but
undergoes transamination with oxaloacetate to give aspartate and 2-oxoglutarate.
The amino group of aspartate is then transferred to hypoxanthine (the constituent of IMP,
inosine-P), and the product (adenosine-P, AMP) adenylate is deaminated by adenylate
deaminase.
NH3
N
N
N
N
OH
Rib P
N
N
N
N
NH2
Rib P
H2O
Aspartate is also the source
of one of the two nitrogen
atoms of urea!
24
In eight proteinogenic amino acids, transamination or oxidative deamination
is not the usual first step in their degradation:
Serine and threonine are deaminated by the action of serine dehydratase,
histidine undergoes deamination by desaturation –
both reactions were already mentioned previously.
In five remaining amino acids, only some of their catabolites is deaminated.
Arginine – deamination occurs after transformation to ornithin,
lysine – after transformation to α-aminoadipate,
methionine – deamination of homoserine,
proline – deamination after conversion to glutamate,
tryptophan – after its transformation to kynurenine, alanine is released.
1 Direct deamination of glutamate (glutamate dehydrogenase reaction).
2 Deamination of aspartate within the "purine nucleotide cycle".
3 Aerobic glycine-cleavage system.
4 Direct deamination of histidine by desaturation.
From the quantitative point of view, the most important reactions which
produce ammonium ion NH4
+
(and molecules NH3) from the α-amino
groups of amino acids within the cells are:
25
Transport of "ammonia" in the blood
Ammonia is a weak base, molecules NH3 are mostly protonated to ammonium ion NH4
+
at physiological pH values. Therefore, we mean both NH4
+
and NH3 when speaking of
ammonia.
Ammonia is toxic, particularly to the brain. Regardless of the intensive
amino acid turnover in the body and high normal concetrations of ammonia
(about 500 μmol / l) in organs other than brain, the concentration of ammonia
in the peripheral blood is quite low, up to 40 μmol / l.
The major transport form of ammonia between tissues is non-toxic glutamine
(concentration in blood plasma 400 – 700 μmol / l).
An additional form of amino groups transfer from peripheral tissues, above all
skeletal muscles, to the liver is alanine (the glucose-alanine cycle).
Alanine concentration in blood plasma is 300 – 400 μmol / l.
Synthesis of glutamine
Glutamine synthetase
GlutamineGlutamate
COOH
H2N–CH
COOH
CH2
CH2
COOH
H2N–CH
CO–NH2
CH2
CH2
The hydrolysis of glutamine
to glutamate is catalysed by
glutaminase.
26
The transfer of –NH2 from muscle
to liver is provided mainly by the
glucose-alanine cycle which is
analogous to the glucose-lactate
(Cori) cycle..
Glutamine is a major energy nutrient for
small intestine mucosa. The nitrogen
atoms of –NH2 are transferred to liver
as NH4
+
(its concentration in portal blood
is very high) and alanine.
Oxaloacetate
Pyruvate
GLUTAMINEALANINE
Glucose
Liver
Muscle
Kidney
Amino acids
Pyruvate
UREA
ALANINE
Portal blood
Urinary NH4
+
Alanine
Central nervous system
Amino acids
Intestinal mucosa
Glutamine
Glutamate
2-Oxoglutarate → CO2
Oxaloacetate
Pyruvate
Bacterial deaminations
NH4
+
Glutamine
2-Oxoglutarate→ CO2
Pyruvate
Glutamine
Glutamate
ALANINE
(depends on ABR)
Glucose
27
The fate of ammonium ion NH4
+
in vertebrates
NH4
+
formed in the breakdown of amino acids
NH4
+
Uric acid Urea
O=C
NH2
NH2
Birds
Snakes
Lizards
Uricotelic animals
Most terrestial vertebrates
Sharks
Adult frogs
Ureotelic animals
Most aquatic vertebrates
(bony fishes)
The larvae of ampihibia
Ammonotelic animals
The ureosynthetic cycle
In most terrestial vertebrates, the liver cells detoxify NH4
+
ions originating
in the catabolism of amino acids by the synthesis of urea.
NH2
NH2
C=O
Urea
Urea (carbamide, carbonic acid diamide) is a non-electrolyte,
highly soluble in water. It diffuses easily across biological
membrane, and therefore it is evenly distributed in all biological
fluids and excreted into urine..
The ureosynthesis is one of the unique functions of the liver cells. These
cells také up glutamine and ammonium ions from the blood, they can also
deaminate glutamate or other amino acids.
The carbon atom of urea is derived from hydrogencarbonate anion, one of
the nitrogens comes from ammonium ion, the other is obtained from
aspartate.
Ureosynthesis is an endergonic process – 3 ATPs are hydrolysed to
2 ADPs, 1 AMP and 4 Pi in the synthesis of one molecule of urea.
It is recommendable to remember, from the physiological standpoint, that the ureosynthesis
is a proton-producing process which consumpts hydrogencarbonate, a buffer base.
29
ATP
The simplified pathway of ureosynthesis
(the ornithine or Krebs-Henseleit cycle)
30
Urea is synthesized in five steps:
1 Synthesis of carbamoyl phosphate in the mitochondrial matrix
The reaction is catalysed by carbamoyl phosphate synthetase I which requires
essentially the allosteric activator – N-acetylglutamate.
The cytosolic form of the enzyme – carbamoyl phosphate synthetase II – utilizes glutamine
instead of ammonia and catalyses the first step of the pyrimidine base synthesis.
2 ATP 2 ADP + Pi
HCO3
–
+ NH4
+
H2N–C
O
O–P–O–
O
O–
Carbamoyl phosphate
(a mixed anhydride,
a high-energy compound)
31
2 The transfer of carbamoyl to ornithine
Carbamoyl phosphate
H2N–C
O
O–P–O–
O
O–
Ornithine
COOH
CH–NH2
CH2
CH2
CH2–NH2
+
Hydrogenphosphate
Citrulline
COOH
CH–NH2
CH2
CH2
CH2–NH
C=O
H2N
HPO4
2–
+
The reaction is catalysed by ornithine transcarbamoylase to form
citrulline.
Both ornithine and citrulline are amino acids, but they are not used as building
blocks of proteins.
The next three reaction of the cycle také place in the cytoplasma.
32
3 The transport of citrulline to the cytoplasma
and condensation with aspartate
COOH
CH–NH2
CH2
CH2
CH2–NH
C=O
H2N
Citrulline Aspartate
COOH
CH2
CHH2N–
COOH
AMP + 2 H2O + 2 P2
ATP
Argininosuccinate
COOH
CH–NH2
CH2
CH2
CH2–NH
C=N–
H2N
COOH
CH2
CH
COOH
The reversible reaction is catalysed by argininosuccinate synthetase and
is driven by the cleavage of ATP into AMP and diphosphate and by the
subsequent hydrolysis of diphosphate.
33
4 The cleavage of argininosuccinate into arginine and fumarate
Argininosuccinate
COOH
CH–NH2
CH2
CH2
CH2–NH
C=N–
H2N
COOH
CH2
CH
COOH
Arginine
COOH
CH–NH2
CH2
CH2
CH2–NH
C=NH2
+
H2N
Fumarate
+
HOOC
CH
CH
COOH
Argininosuccinase cleaves argininosuccinate into arginine and fumarate.
The carbon skeleton of aspartate is preserved in the form of fumarate.
The synthesis of fumarate by the urea cycle is important because
it links the urea cycle and the citric acid cycle: Fumarate after hydratation to
malate may enter mitochondrion and be oxidized to oxaloacetate, which can
undergo transamination to aspartate. .
34
5 The hydrolysis of arginine generates urea and ornithine
Arginase
Urea
NH2
NH2
C=O
Ornithine
COOH
CH–NH2
CH2
CH2
CH2–NH2
COOH
CH–NH2
CH2
CH2
CH2–NH
C=NH2
+
H2N
Arginine
H2O +
35