Download Title: Author - Department of Biochemistry and Molecular Biology

Title:
Study of transaminases
Author:
János András Mótyán, Ph.D.
assistant lecturer
Department of Biochemistry and Molecular Biology
Faculty of Medicine, University of Debrecen
2015
Date: 2014.12.01-2015.01.31.
The development of this curriculum was sponsored by TÁMOP 4.1.1.C-13/1/KONV-2014-0001.
The project is supported by the European Union and co-financed by the European Social Fund.
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THEORETICAL BACKGROUND
1. TRANSAMINASES
The Study of transaminases practice includes the study of the reversibility of transaminase
reaction, the determination of serum glutamate-oxaloacetate-transaminase (GOT) and glutamatepyruvate-transaminase (GPT) activities by optical test and the determination amino acid
composition of serum and urine in case of a metabolic disease (phenylketonuria).
Therefore, here we are focusing on the transamination reactions and the degradation of
phenylalanine and tyrosine, and we describe the theoretical background of the experiments.
1.1 Introduction
Human proteins are built up by 20 different amino acids (Figure 1). These amino acids
commonly found in proteins as residues have L-configuration (except glycine) and are α-amino
acids (except proline, which is imino acid). All of these amino acid contains a carboxyl-group and
an amino group attached to the α-carbon atom (α-amino acids), furthermore, a distinctive side
chain. Amino acids can be differentiated based on the characteristics of the side chains: they can
be classified into nonpolar, aromatic, polar and uncharged, negatively or positively charged
groups.
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Figure 1. Building blocks of amino acids.
Several common types of reactions are involved in the metabolism of the amino acids,
which have key role both in their degradation and in their synthesis. Such reactions are the
transamination, deamination (oxidative and non-oxidative), decarboxylation (oxidative and nonoxidative), monooxigenation, dioxigenations and the transfer reactions of one-carbon units.
Enzymes are classified into 6 classes by the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (NC-IUBMB) based on the type of reaction
catalyzed:
1. oxidoreductases
2. transferases
3. hydrolases
4. lyases
5. isomerases
6. ligases
The transferases belong to the 2nd enzyme class (EC 2) and catalyze the transfer of a
functional group between the donor and the acceptor molecule. The group of enzymes transferring
nitrogenous groups (EC 2.6) contains the group of aminotransferases, also referred as
transaminases (EC 2.6.1), which enzymes catalyze the transfer of an amino group from an amino
acid to a keto acid. In this reaction a new amino acid and a new keto acid is released.
1.2 The transamination reactions
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The role of transamination reactions is the elimination of the amino nitrogen from the
amino acids. While in case of the deamination reaction the nitrogen is removed from the amino
acid pool, the transamination reactions do not eliminate nitrogen from the pool. Quantitatively
transamination it is the most important reaction type of the amino acid metabolsim. Transaminases
exist for all amino acids except Lys and Thr. Usually the transamination is the starting reaction
during the degradation of amino acids. Transaminases can be classified based on their cellular
localization (cytoplasmic or mitochondrial), the position of the amino group (α, β, etc.) or the
substrate partner molecule.
In the transamination reaction the amino group of the amino acid substrate is transferred to
pyridoxale phosphate cofactor of transaminase enzyme followed by its transfer to the keto acid
substrate. Keto acid and amino acid products are produced in the reaction.
Transaminase catalyzed reaction is reversible. Pyridoxale phosphate cofactor is regenerated
in the reciprocal reaction, in which keto acid is used as substrate and amino acid product is
released (Figure 2).
Figure 2. General scheme of transamination. PLP = pyridoxale phosphate, PMP = pyridoxamine
phosphate.
Pyridoxale phosphate (PLP) is used as cofactor (Figure 3) by all transaminases. Pyridoxale
phosphate is a derivative of Vitamin B6.
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Figure 3. Structure of pyridoxine and pyridoxale phosphate cofactors.
1.2.1. Mechanism of transamination
The pyridoxale phosphate is attached to a lysil sidechain of the enzyme via a Schiff base
(imine) linkage, which could be substituted by an amino acid. The pyridoxale phosphate cofactor
is covalently attached to the enzyme. After the binding of a substrate the cofactor will be attached
to the amino acid, the covalent linkage will be disrupted between the cofactor and the enzyme.
There are non-covalent interactions between the enzyme and the amino acid-bound cofactor.
In the first step of the transamination reaction the amino group of the amino acid substrate
attacks the enzyme-cofactor Schiff base, which step is resulted in the formation of an amino acidPLP aldimine. Through a quinonoid intermediate state the Schiff base is hydrolyzed, a
pyridoxamine phosphate and a keto acid product is released (Figure 4). Figure 4. shows the first
part of the overall catalytic process catalyzed by aminotransferases, in which amino acid substrate
enters the catalytic cycle and keto acid product is released (left to right). To regenerate the PLPenzyme, the “first” keto acid (released as product) is replaced by a “second” keto acid which is
used as substrate in the reversal steps (right to left). The amino acid is transferred from the
pyridoxal phosphate to the “second” keto acid, leading to the release of an amino acid product.
Figure 4. Mechanism of transamination.
1.2.2. Relevance of transamination
1.2.2.1 Transport of reduced electron carriers into the mitochondria from the cytosol
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Reduced electron carriers are oxidized in the terminal oxidation. The reduced carriers
which were produced by the pyruvate dehydrogenase or by reactions of citric acid cycle could be
oxidized within mitochondria. The NADH released in a glycolytic reaction (catalyzed by
glyceraldehide-3-phosphate-dehidrogenase) requires a transport mechanism, because the inner
mitochondrial membrane is not permeable for NAD+ and NADH+H+.
There are two main transport mechanisms which are responsible for the mitochondrial
transport of NADH produced in the cytosol. These are the malate-aspartate and the
glicerophosphate shuttles. Both shuttles are able for the transport of the reduced electron. While
the malate-aspartate shuttle is reversible and works in case of high cytosolic NADH concentration,
the glycerophosphate shuttle is irreversible and is independent from the NADH concentration.
In case of the glycerophosphate shuttle the glycerol-3-phosphate-dehidrogenase converts
the dihydroxiacetone-phosphate to glycerol-3-phosphate in the cytosol, along with the oxidation of
NADH. After entering the mitochondrial matrix, the mitochondrial isoform of the enzyme
converts the glycerol-3-phosphate back to dihydroxiacetone-phosphate along with the reduction of
its FAD cofactor. By the help of this shuttle mechanism, the NADH released in the cytosol reaches
the mitochondrial respiratory chain as FADH2.
The malate-aspartate shuttle is also responsible for the transport of reduced electron
carriers through the inner mitochondrial membrane. By the help of this shuttle mechanism, the
NADH released in the cytosol reaches the mitochondrial respiratory chain as NADH. In the first
step of the transport mechanism the NADH reduces the oxaloacetate to malate in the
intermembrane space, which reaction is catalyzed by the malate-dehydrogenase enzyme. Malate
enters the matrix by the malate-α-ketoglutarate transporter, where it is converted back to
oxaloacetate, along with the reduction of NAD cofactor of malate-dehydrogenase and the release
of NADH. In a transamination reaction the oxaloacetate is converted to aspartate (while glutamate
is converted to α-ketoglutarate) by the aspartate-aminotransferase (glutamate-oxaloacetate
transaminase). Aspartate is transported into the intermembrane space by the glutamate-aspartate
transporter, where α-ketoglutarate is converted to oxaloacetate in a repeated transamination
reaction. Oxaloacetate could be reduced by the malate dehydrogenase and enter the cycle again to
transport NADH into the mitochondrial matrix (Figure 5).
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Figure 5. The malate-aspartate shuttle.
1.2.2.2 Funneling nitrogen into glutamate
The role of transamination is the funneling of amino nitrogens from amino acids to
glutamate. The glutamate could be deaminated (see point 3) and the nitrogen funnelled into
glutamate could be used to convert it to urea and eliminate from the amino acid pool (see point 5).
The glutamate dehydrogenase catalyzed reaction has the highest relevance in the deamination.
Deaminated amino acids can be used to produce energy by their fully oxidation.
The glutamate has great relevance in transamination reactions, because the glutamate-αketoglutarate amino acid-keto acid pair, which are involved in several transamination reactions.
1.2.2.3 Trans-deaminastion
In case of trans-deaminastion the common intermediate is glutamate, which is further
converted by oxidative deamination after a transamination reaction. α-Ketoglutarate and ammonia
is released in the reaction catalyzed by glutamate dehydrogenase (GDH) (Figure 6). This
oxidative step uses NAD cofactor and due to the release of ammonia the nitrogen is eliminated
from the amino acid pool.
The glutamate dehydrogenase catalyzed reaction is reversible in vitro and is shifted
towards the production of ammonia in vivo. In the reversed reaction glutamate is released and
NADPH is used as cofactor.
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Figure 6. Trans-deamination.
1.2.2.4 The glucose-alanine cycle
Glucose-alanine cycle has key role in the amino acid transport between different tissues.
This cycle works between the liver and the muscle. The glucose-alanine cycle provides substrate
for the muscle by the help of gluconeogenesis. Muscle has high transamination capacity, because
the pyruvate (produced by the glycolysis) is converted to alanine by transamination. Produced
alanine is transported to the liver by the bloodstream, where it is converted back to pyruvate by a
repeated transamination reaction. Pyruvate is a substrate for gluconeogenesis, and its conversion to
glucose provides energy source for the muscle, it is transported to the muscle by the bloodstream
(Figure 7).
However, NADH is released during the conversion of glucose to pyruvate, this NADH is
not used for the conversion of pyruvate to lactate (in case of normoxia), because this reaction
would deplete the pyruvate and the pyruvate would not be able to enter the transamination reaction
and would not be converted alanine. In case of relative hypoxia (for example in case of extensive
physical activity) the tissue does not completely oxidize glucose to water and CO2, therefore,
elevated amount of lactate is produced due to the anaerob glycolysis.
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Figure 7. The glucose-alanine cycle.
1.2.2.5 Urea synthesis
Nitrogen is provided for the urea synthesis not only by deamination reactions but by
transamination reactions. In this way both nitrogens of urea could come from glutamate amino
nitrogen. The aspartate required for urea synthesis is released by a transamination reaction. The
ammonia released in the glutamate dehydrogenase (GDH) catalyzed reaction is used in the
preparatory step of urea cycle by carbamoyl phosphate synthase I (CPS I) for the synthesis of
carbamoyl-phosphate. The aspartate entering the urea cycle is produced by the conversion of
oxaloacetate to glutamate (transaminase reaction), therefore, there is a strong connection between
the urea cycle and citric acid cycle (Figure 8).
Figure 8. Connection between urea cycle and citric acid cycle.
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1.2.2.6 Conversions of amino acids
Commonly the degradation of amino acids is started with transamination, in which reaction
the glutamate-α-ketoglutarate amino acid-keto acid pair is involved. With the exception of lysine
and threonine transaminases exists for all amino acids.
If it is necessary, the synthesis of non-essential amino acids can be carried out by
aminotransferase catalyzed reactions. In this case an α-keto acid is used as precursor and an amino
group is transferred. The transaminase reactions of essential amino acids have only one direction,
because the equivalent α-keto acids cannot be synthesized in humans.
1.2.3 Clinical correlations
The number of metabolic diseases caused by of transaminase deficiencies is small, possibly
beause the complete lack of transaminase activity is not compatible with life.
For example, degradation of valine and isoleucine starts with transamination. Enzymopathies of these enzymes are known and lead to hypervalinaemia, hyperleucine-isoleucinaemia.
Transaminases have high diagnostic value, because the serum level of transaminases gives
important information from the viewpoint of diagnosis. Transaminases are intracellular enzymes,
therefore, their concentration in serum is low.
Normal value of glutamate-oxaloacetate-transaminase (GOT) enzyme in serum is 60-260
nkat/l (3.8-15.8 U/l), while normal value of glutamate-pyruvate-transaminase (GPT) is 8-290
nkat/l (0.5-17.3 U/l). Tissue lesions (e.g. cell necrosis, changes of membrame permeability, etc.)
lead to increase in serum concentration of the enzymes.
Heart muscle tissue contains highest amount of GOT, while liver contains highest amount
of GPT. Measurement of transaminases is important in the diagnosis of injury of heart muscle or
liver. Serum GOT activity increases 4-6 hours after heart infraction, reaches the maximum after
~24 hours (could reach 1700 nkat/l). This value decreases until it reaches the normal value, which
takes approximately 7 days. During this period the GPT activity is constant. Serum GPT activity
increases mainly after the damage of liver cells, high serum level can be measured in case of
infectious liver lesions. Increased GPT activity can be measured after heart infraction due to the
impaired blood supply and damage of liver cells.
1.3 Reactions of amino acids: phenylalanine and tyrosine
Degradation of phenylalanine and tyrosine occurs mainly in the liver.
The major pathway of the degradation is started with a monooxigenase reaction leading to
the conversion of phenylalanine to tyrosine (Figure 9).
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There is a minor pathway of the phenylalanine degradation starting with a transamination
reaction, but the capacity of this enzyme is low. Minor pathways have relevance in case of high
phenylalanine concentration. In this case the phenylalanine is converted to phenylpyruvate by
transamination. Further minor pathway is the conversion to phenyllactate or phenylacetate (see
later) (Figure 11).
Phenylalanine hydroxylase catalyzed reaction is monooxigenation, where one atom of
molecular oxygen forms hydroxyl group of the substrate while the other forms water with the
hydrogens of the cofactor. This reaction releases tyrosine. From this step the phenylalanine and
tyrosine has the same metabolism (Figure 9).
Figure 9. Conversion of phenylalanine and tyrosine to homogentisate.
The phenylalanine hydroxylase catalyzed monooxigenation requires oxigent and
tetrahydrobiopterin (BH4) as cofactor. Tetrahydrobiopterin is synthesized from GTP precursor by
GTP cyclohydrolase I. BH4 is oxidized to dihydrobiopterin (BH2) in the phenylalanine
hydroxylase catalyzed reaction. BH2 could be reduced to BH4
by
dihydrobiopterin reductase
(Figure 10).
Figure 10. Reaction catalyzed by dihydrobiopterin reductase.
Second step of the major phenylalanine and tyrosine degradation pathway is the conversion of
tyrosine to p-hydroxyphenylpyruvate, together with the conversion of α-ketoglutarate to glutamate
(Figure 9). The p-hydroxyphenylpyruvate is converted to homogentisate by the dyoxigenase
enzyme. In the following steps acetoacetate and fumarate is produced from homogentisate.
1.3.1 Phenylketonuria and possible causes
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Phenylalanine and tyrosine have several precursor functions. They are important precursors
of the neurotransmitter (catecholamines: dopamine, norepinephrine, epinephrine) and thyroxine
synthesis. Tyrosinase converts tyrosine to dopa, which product is used in melanine (pigment)
synthesis.
The most common disease which affects the amino acid metabolism is phenylketonuria.
Deficiencies affecting the metabolism of phenylalanine and tyrosine lead to the development of
different metabolic disorders, for example the impaired tyrosine synthesis leads to reduced
catecholamine synthesis and melanine production.
1.3.1.1 Classical phenylketonuria - phenylalanine hydroxylase deficiency
The consequence of mutation of the phenylalanine hydroxylase gene is the phenylketonuria
(PKU). It is an inherited autosomal recessive disorder in which phenylalanine-tyrosine conversion
is impaired. The consequence of the enzyme deficiency is the accumulation of the phenylalanine,
because it cannot be converted tyrosine. Deficiency of phenylalanine hydroxylase enzyme leads
classical phenylketonuria.
PKU can be characterized by high serum concentration of phenylalanine. Main symptom of the
disease is the lighter skin color due to the impaired melanine synthesis and mental retardation.
Early diagnosis of PKU is very important, because in case of the recognized PKU a strict diet
could be applied, which contains sufficient amount of essential amino acids and very low
concentration of phenylalanine. This therapy can be applied efficiently, and in this way the
development of serious symptoms (including mental retardation) can be avoided. Phenylketonuria
screening of newborn babies is compulsory in Hungary.
1.3.1.2 Cofactor deficiency - dihydrobiopterin reductase deficiency
In contrast with classical phenylketonuria, 3% of PKU cases is caused by cofactor
deficiency. In the case of cofactor deficiency the phenylalanine hydroxylase reaction is affected,
and phenylalanine cannot be converted to tyrosine, because this monooxigenation reaction requires
tetrahydrobiopterin cofactor. The accumulated phenylalanine is converted phenylpyruvate by a
transaminase (along with the conversion of pyruvate to alanine) (Figure 11).
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Figure 11. Production of phenylpyruvate, phenyllactate and phenylacetate.
Phenylpyruvate can be further converted to phenyllactate ot phenylacetate (Figure 11),
their accumulation could affect the central nervous system and causes severe symptoms. These
molecules lead to the loss of the myelin coat of neurons causing mental retardation in case of
cofactor deficiency, as well.
The cofactor deficiency could affect not only the phenylalanine degradation but other synthetic
pathways (e.g. dopamine). Urine sample of healthy persons contain only small amount of these
metabolites, but high concentration can be detected in the urine of PKU patients. Characteristic
symptom of this disease is the mousy odor of urine.
Phenylalanine is toxic for neurons and this effect is responsible for the mental retardation,
furthermore, the aromatic amino acid uptake of neurons is inhibited due to the high phenylalanine
concentration. In the case of cofactor deficiency the therapy also involves synthetic diet low in
phenylalanine. In case of biopterin deficiency treatment includes the administration of biopterin.
Deficiency of reductase causes more severe symptoms, the impaired catecholamine and serotonin
synthesis is responsible for the neurological symptoms.
1.3.1.3 Maternal phenylketonuria
Even if the neonate is a heterozygote, high phenylalanine concentration in the mother's blood
causes severe mental retardation and heart problems of the fetus.
1.3.1.4 Tyrosine aminotransferase deficiency
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Rare but severe disease. Tyrosinaemia and mental retardation. Besides the neurological symptoms
the eye is also affected, skin lesions also develop. The “cabbage" odor smell is characteristic for
this disease.
1.3.1.5 Enzyme deficiencies of the BH4 synthesis
In very rare cases the synthesis of the cofactor from GTP may also be affected due to the
deficiency of one of the enzymes catalyzing the process.
1.4. Theoretical background of practices:
1.4.1 Study of reversibility of transaminase-reaction
Amino acids are detected using ninhydrin reaction. Ninhydrin reaction is positive for every αamino acid. Ninhydrin reagent reacts with the terminal amino group, followed by the condensation
of the ammonia (released from α-amino acids) with the ninhydrin, and a purple coloured product is
formed (Figure 12).
Figure 12. Detection of amino acids by ninhydrin reaction.
1.4.2 Determination of serum GOT and GPT activity by optical test
Wartburg optical test is used in the practice to determine the serum activity of glutamateoxaloacetate-transaminase (GOT) and glutamate-pyruvate-transaminase (GPT) aktivitásának
(Figure 13).
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Figure 13. GOT and GPT catalyzed reactions.
The Warburg optical test was described by Warburg at 1936. This colorimetric
measurement is based on the different absorption properties of NAD+ (NADP+) and NADH
(NADPH). The change of the NADH or NADPH concentration can be measured at 366 nm
wavelength by this colorimetric method, because only the reduced forms of the cofactors absorb,
the oxidized forms hardly show any absorbance around this wavelength (Figure 14). Change of
the absorbance is measured in a 340-366 nm wavelength range, therefore, the test is referred as
UV-test. This test is a useful method for enzyme activity measurements.
Figure 14. Absorption of NAD+ (NADP+) and NADH (NADPH) at different wavelengths.
This UV-test is used in the practice to determine GOT and GPT activities. But the activities
of these enzymes cannot be determined directly, because there is no net NAD+ or NADH release
or production in the catalyzed reaction. A coupled optical test must to be applied for the activity
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measurement, which means that the transaminase reaction (reaction of interest) must to be
completed with a second reaction (indicator reaction), in which NADH (or NADPH) is used or
released by an auxiliary enzyme.
In the GOT catalyzed reaction aspartate and α-ketoglutarate substrates are converted to
glutamate and oxaloacetate products. This reaction is followed by the conversion of oxaloacetate
to malate in the malate dehydrogenase (MDH) auxiliary enzyme catalyzed indicator reaction,
followed by the release of NAD+ (Figure 15). The decrease of the absorbance is measured,
because the MDH uses NADH, therefore, decrease of NADH concentration leads to decrease of
absorbance measured at 366 nm.
Based on the Lambert-Beer law the absorbance can be used to calculate the concentration
of molecule absorbing light.
A=Ɛ*c*l
A
Ɛ
c
l
- absorbance
- molar extinction coefficient
- concentration of molecule absorbing light
- length of way of light
Figure 15. Reactions of coupled optical test used for GOT activity measurement.
In the GPT catalyzed reaction alanine and α-ketoglutarate substrates are converted to glutamate
and pyruvate products. This reaction is followed by the conversion of pyruvate to lactate in the
lactate dehydrogenase (MDH) catalyzed indicator reaction, followed by the release of NAD+
(Figure 16).
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Figure 16. Reactions of coupled optical test used for GPT activity measurement.
To make the reaction of interest to be the rate-limiting step of the coupled reaction, the
excess of MDH and LDH auxiliary enzymes must to applied. The enzyme activities can be
calculated based on the change of the absorbance. One katal is the amount of enzyme that converts
1 mol substrate per second, while one unit (U) is the amount of enzyme that catalyzes the reaction
of 1 µmol substrate per minute.
1.4.3 Study of reactions of amino acid: phenylketonuria. Determination of amino acid
composition of serum and urine by thin-layer ion-exchange chromatography
Thin-layer chromatography is a simple and fast analytical method, which is useful to examine
amino acids, short peptides, antibiotics, etc. Therefore, this method can be applied to determine the
amino acid composition of serum and urine. Thin-layer chromatography is used in the laboratory
practice to study a metabolic disease.
The Polygram (Ionex-25 SA-Na) is a cation-exchanger thin-layer carried by plastic foil.
The ion exchange resin is prepared from polyacrylamide carrying sulfate groups, the counter ions
are Na+. The effectiveness of the separation of amino acids depends on the pH and ionic strength
of the buffer system. We use sodium citrate buffer, pH 5.28, which separates basic and aromatic
amino acids effectively. The amino acids are separated according to their positive charges
depending on the number and pK value of their -NH groups.
Use micropipette to apply the samples onto the thin-layer plate. Use pencil to indicate a start-line
approximately 1 cm from the bottom of the plate. Apply the samples onto the start line. Use hair
dryer during this process, to make as small sample spots as it is possible to avoid the contact of the
spots and the mixture of samples. After drying the spots place the thin-layer plate into the
chromatography tank containing developing buffer. Do not let the sample spots to sink into the
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buffer! The buffer will run on the plate due to the capillary effect. Let the buffer run to 1 cm from
the top of the plate, after it the plate need to be dried again by hair dryer. To develop the colors,
the plate need to be sprayed by the ninhydrin reagents and dried again by hair dryer. Results can be
evaluated based on the running distance of the sample spot, if it is necessary, the diameter of spots
can be also determined.
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Self-control questions
1. How transamination reactions are involved in the mitochondrial transport of reduced electron
carriers produced in the cytosol?
2. How transamination reactions are involved in the glucose-alanine cycle?
3. What would be your diagnosis if the serum concentration of GOT (glutamate-oxaloacetatetransaminase) would be higher then 260 nkat/l?
4. What would be your diagnosis if the serum concentration of GPT (glutamate-oxaloacetatetransaminase) would be higher then 290 nkat/l?
5. What is the difference between the classical phenylketonuria and the cofactor deficiency?
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URL: http://www.chem.qmul.ac.uk/iubmb/enzyme
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