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EXAMINATION QUESTIONS BIOCHEMISTRY

Structure of hemoglobin, structure-function relationships (the oxygen
saturation curve, inducement of hemoglobin saturation and oxygen
transport. Bohr effect):
Structure of hemoglobin:
Hemoglobin is a tetrameric protein of erythrocytes that transports O
2
to tissues and returns
CO
2
and protons to the lungs. Myoglobin is a monomeric protein of red muscles that stores
oxygen as a reserve against oxygen deprivation (= bervelse).
Hemoglobin and myoglobin contain heme that is a cyclic tetrapyrrole and a chelate of
protoporphyrin IX and Fe
2+
, which has 4 molecules of pyrrole linked by -metylene
bridges. The reason for the red color of heme is that its planar structure absorbs the red
component of visible light. At the -position of heme there are 4 methyl groups, 2 vinyl
groups, and 2 propionic acids. At the center of the planar tetrapyrrole a Fe
2+
molecule
resides. The biological activity is destroyed when it is oxidized to Fe
3+
.
Hemoglobin has 4 globular protein subunits that each consist of a protein chain and a non-
protein heme group. Each protein chain is arranged into a set of -helical segments connected
together in a globin fold arrangement. This folding forms a pocket that strongly binds the
heme group. The -helical structural segments are termed A-H and contain about 7-20 amino
acids. The exterior part of the hemoglobin is polar and contains polar amino acids. The
interior of the hemoglobin contain non-polar amino acids. The exception is histidine located
at the helices E and F that lie close to the heme iron and function in binding oxygen to the
heme. The proximal histidine His F8 is linked to the 5
th
coordination position of Fe
2+
. The
distal histidine, His E7 is linked to the 6
th
coordination position of Fe
2+
and lies opposite to
the proximal histidine. When oxygen (O
2
, two molecules of oxygen binds) binds to
hemoglobin it binds at the 6
th
coordination position of Fe
2+
between the histidine E7 and the
iron.
Structure-function relationships:
The oxygen saturation curve: The oxygen saturation curve is associated to the dissociation
of hemoglobin that states the percentage of saturation (%) to the gaseous pressure of oxygen
(mmHg).
The oxygen binding curve for myoglobin is hyperbolic. It readily binds oxygen to its only
heme group but it releases only a small fraction of its oxygen. This explains why it is
unsuitable as an oxygen deliverer and better for oxygen storing.
The oxygen binding curve for hemoglobin has a sigmoidal shape. Each of the four heme
groups contained in hemoglobin can bind one O
2
atom and thus in total four such oxygen
atoms can be bound to one molecule of hemoglobin. The reason for the sigmoidal shape of the

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saturation curve is due to the cooperative effect that is an inducement of binding oxygen
between the four globin chains (oxygenated hemoglobin). When one heme of a globin chain
has bonded to one molecule of oxygen it gets easier for the second and then third heme groups
of other globin chains to bind oxygen. The plateau part of the curve corresponds to the partial
pressure and saturation in pulmonary capillaries and the steep part corresponds to the
conditions of the systemic capillaries. The reason for this cooperative effect is that after
binding of one oxygen molecule to a heme group fewer salt bridges have to be ruptured to
bind more oxygen. P50 states the partial pressure of oxygen that half-saturates hemoglobin
and it has different characteristic values of different types of hemoglobin.
Bohr effect:
When oxygen binds to hemoglobin it makes the ion, the His F8 and its bonded residues move
towards the plane of the heme ring. This motion causes ionic interactions (salt bridges, COO
-

and H
+
mainly from glutamate and aspartate) to be ruptured (hydrophobic interactions
remain). When the salt bridges are ruptured protons arise and these drive the formation of
carbonic acid from bicarbonate. This action triggers the release of O
2
from hemoglobin and
the T-form and salt bridges are reformed. 2,3-Bisphosphoglycerate acts as a stabilizing
agent of the non-oxygenated form of hemoglobin. This conformational change when the
proton concentration is elevated and oxygen is released from hemoglobin is called the Bohr
effect. Conversely, when the pO
2
is increased, hemoglobin will be oxygenated and protons
will be released.
1. Normal haemoglobin types in blood, haemoglobin concentration.
Other forms (glycohaemoglobin, methaemoglobin,
carboxyhaemoglobin) and abnormal haemoglobins.
Normal hemoglobin types in blood:
Hb A (
2

2
): Adult blood 97%
- Hb A
0
(
2

2
) partly HbA-Glc
- Hb A
1
(
2

2
) glycation on terminal NH
2
group of -globin 5%
- Hb A
2
(
2

2
) 2,7 %
Hb F (
2

2
): Fetal blood 0,5%
Hb S: Sickle hemoglobin
Hb M: Methemoglobin
Concentration of Hb in blood: 2,15-2,65 mmol/l (tetramer)
Glycohemoglobin:
When blood glucose enters erythrocytes it glycates the -amino group of lysine and the
amino terminals of hemoglobin. Since the half-life of an erythrocyte of 60 days , the level of

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glycated hemoglobin reflects the mean blood glucose concentration over the past 6-7 weeks
and this provides valuable information in diabetes controls.
Methemoglobin:
When the ion of heme Fe
2+
is oxidized, methemoglobin Fe
3+
is formed. This oxidized form of
iron cannot bind or release oxygen. Methemoglobin can arise from hemoglobin M
(Methemoglobinemia), reduced activity of methemoglobin reductase or as a side effect of
agents such as sulfonamides. In hemoglobin M the histidine F8 has been replaced by
tyrosine that stabilizes the Fe
3+
form. In the -chain the T-state is favored and the Bohr effect
is absent. In the -chain there is a switching between T- and R state and here the Bohr effect
is present.
Carboxyhemoglobin:
Carboxyhemoglobin is formed when carbon monoxide is inhaled and bonded to the heme
groups of hemoglobin in erythrocytes. The binding of carbon monoxide hinders the binding of
oxygen and it can lead to suffocation if not oxygen is quickly administered. Oxygen-heme is a
stronger bond than that is CO-heme since the His E7 makes the binding angle of CO-heme
less favorable than that of O
2
-heme.
Abnormal hemoglobins:
Hemoglobin S: It is caused by a point mutation where the amino acid glutamic acid has been
replace by the non-polar amino acid valine on the -globin chains. Hydrophobic regions of
these chains generate sticky pads of both the oxygenated and deoxygenated forms of
hemoglobin S. The T-form of hemoglobin A matches the sticky pads of the T-form of
hemoglobin S that together create misshaped fibrous erythrocytes that can cluster and cause
thrombosis and that hardly undergo lysis due to their rigid shape. The polymerization of the
fibrous structure can only be terminated with a Hb A in the T-form that is unable to form
sticky pads.
Thalassemias: a group of genetic defects where there is a partial or total absence of - or -
chains of hemoglobin. Depending on which chain is absent, - and thalassemias are
distinguished.
2. Enzymes - structure and catalytic function, characteristics of
biocatalysis. Enzyme-substrate interaction, examples of mechanisms
of enzyme-catalyzed reactions. The term isoenzymes:
Structure and catalytic function:
Enzymes enhance the reaction rate of a reaction without being consumed themselves. They
are very substrate- and reaction specific. Similar substrates may be catalyzed by some
enzymes. Enzymes are also stereo-specific and typically catalyze only one stereoisomer of a

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given compound. Enzymes are sequences of amino acids that are genetically encoded by
DNA of each individual.

Enzymes are grouped into 6 classes:
1. Oxidoreductases: catalyze oxidations and reductions
ethanol + NAD
+
acetaldehyde + NADH + H
+

2. Transferases: catalyze transfer of moieties (= delar) such as glycosyl, methyl and
phosphoryl groups
glucose + ATP glucose 6-phosphate + ADP

3. Hydrolases: catalyze hydrolytic cleavage of C-C, C-O, C-N and other bonds
glucose 6-P + H
2
O glucose + P
i


4. Lyases: catalyze cleavage of C-C, C-O, C-N and other bonds by atom elimination,
leaving double bonds
Fumarate + H
2
O L-malate

5. Isomerases: catalyze geometric or structural changes within a molecule
UDP-glucose UDP-galactose

6. Ligases: catalyze the joining together of two molecules coupled to the hydrolysis of
ATP
Pyruvate + CO
2
+ ATP + H
2
O oxaloacetate + ADP + P
i

Many enzymes contain non-protein molecules that are divided into cofactors, coenzymes,
and prosthetic groups. Many of these groups are derivatives of B vitamins.
Prosthetic group: the prosthetic group forms a tight and stable incorporation in the enzyme
by either covalent- or non-covalent bonds. Many metals are prosthetic groups.
Examples: Pyridoxal phosphate, FMN, FAD, thiamin pyrophosphate, biotin, Co, Cu, Mg,
Mn, and Zn.
Cofactors: Cofactors, unlike prosthetic groups, bind to the enzyme, or a substrate such as
ATP, in a dissociable and transient manner. Therefore, cofactors need to be present in the
nearby area of the reaction. Most metals are also cofactors. Enzymes that require metal-
cofactors are called metal-activated enzymes, while enzymes requiring metal-prosthetic
groups are called metalloenzymes.
Examples: NAD, NADP
Coenzymes: Coenzymes serve as recyclable shuttle that transport substrates from the place
they are generated to the place where they are utilized. They also stabilize reactive substrates

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such as H
+
and OH
-
that are unstable in aqueous environment of the cell.
Examples: Methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides
(dolichol).
Biocatalysts are natural catalysts, e.g. protein enzymes that perform chemical transformations
on organic compounds.
Enzyme-substrate interactions:
Enzymes form enzyme-substrate complexes (ES) at the active site of the enzyme. The ES
complex has higher stability than that of the enzyme itself alone. The active site is a pocket on
the surface of the enzyme, or at the interface of subunits of the enzyme where catalysis of a
reaction takes place. It is highly specific with certain amino acid sequences that match a
specific substrate. In the active site give a shielded environment for the reaction to occur and
it also contains cofactors or prosthetic groups needed for the reaction to take place. When the
enzyme binds to the substrate is undergoes conformational changes (the induced fit model).
There are four mechanisms that enzymes use to achieve catalytic enhancement of a reaction:
1. Catalysis by proximity: For molecules to react they must be at a bond-forming
distance to each other. By increasing the concentration of a substrate the probability of
a reaction will increase and by that also the velocity.

2. Acid-base catalysis: Ionizable aminoacyl side chains of prosthetic groups are able to
act as acids or bases by donating H
3
O
+
or OH
-
ions. Depending on if the substrate is
sensitive to the H
3
O
+
/ OH
-
donation of a specific acid/base present we speak of
specific acid or base catalysis. If the substrate is not sensate to any particular acid
or base we speak of general acid- or base catalysis.
Example: Aspartic protease family (pepsin, cathepsin etc.)

3. Catalysis by strain: Enzymes that are involved in breaking a covalent bond (-lytic
reactions) typically bind to their substrate in a conformation that is not as favored for
the bond to be cleaved. This unfavored bonding makes the bond strained and
contributes to its weakening. When the bond is more vulnerable it will be easier for
the enzyme to cleave it.

4. Covalent catalysis: The enzyme forms a covalent bond with one or more substrates
and the modified enzyme then becomes the reactant. Covalent catalysis enables a new
reaction pathway that requires less activation energy that makes it much faster. After
completion of the reaction the enzyme regains its original conformation and shape and
is ready for a new round. Residues on the enzyme that participates in the covalent
bonding are usually serine, cysteine and sometimes histidine.
Examples: Chymotrypsin, Fructose-2,6-bisphosphatase
Sequential reaction = Both substrates bind to the enzyme, chemical conversion proceed and
both products are released.

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Ping-pong effect = One substrate binds, is converted and then released. The same thing
happens then with the second substrate. Typical for aminotransferases.


Isoenzyme:
Isoenzymes are distinct enzyme forms that catalyze the same reaction. They may have
different affinity to the substrate or sensitivity to regulatory factors.
Example: Hexokinase and glucokinase that both catalyze the conversion of glucose to
glucose-6-phosphate.
3. Kinetics of enzyme-catalyzed monomolecular reactions: the term
reaction rate, factors affecting the rates of enzyme-catalyzed
reactions. The progress curves, the Michaelis-Menten plots
(saturation curves), the K
m

value and its significance:

Gibbs free energy G describes both the direction of a reaction and what concentrations the
reactants and substrates will have at equilibrium.
G = G
P
- G
S
= H - ST
If the free energy of formation of the substrate is higher than the formation of the products the
reaction is said to be spontaneous and exergonic. If the free energy of formation of the
products is higher than that of the substrate the reaction is said to be non-spontaneous and
endergonic. However, G is independent of the reaction mechanism and cannot explain the
reaction rate.
All reactions consists of two phases; formation following decay. G = G
F
+ G
D
.
Reactions can have one or several transition states, G represents the sum of all changes in
formation and decay of all the transition states. G
F
for reaching a transition state is referred
to as activation energy. The time it takes to overcome the barrier of activation energy is
determining the time it takes for the reaction to proceed. Enzymes lower the activation barrier
of a reaction. They do this by binding more tightly to the intermediate of the reaction than to
both the substrate and product. Stabilization can involve acid-base groups suitably positioned
to provide protons, suitably positioned charged groups or metal ions that stabilize charges.
Enzymes undergo conformational changes during catalysis, but they always regain their
original composition at the end of the reaction. Enzymes do not change the equilibrium
constant.

There are several factors determining how fast a reaction can proceed:
- Temperature: Increases the reaction rate for both enzyme-catalyzed and uncatalyzed
reactions. By raising the temperature the kinetic energy of the molecules increases and
the number of molecules capable of overcoming the activation energy. Enzymes are
polypeptide chains and start to denature at too high temperatures which accompanies
the loss of catalytic activity.

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- Hydrogen ion concentration: The presence of hydrogen ions stimulates the
formation of salt bridges between the enzyme and substrate. For enzymes involved in
acid-base catalysis the residues involved must be in appropriate state of protonation.
The most common ionizable groups are negatively charged carboxyl groups and
positively charged amines. Different enzymes have different pH optimums depending
on when they have maximal ionized groups that can bind the substrate.
Reaction rate:

Reaction velocity: v = - S / t = P / t (mol/ls)

Enzyme-catalyzed reactions follow the so-called zero order where the reaction rate is
independent of the reactants for a certain period from the start. Enzyme-catalyzed reactions
with high substrate concentrations have enzymes that are fully saturated with the substrate.
The rate of the process is constant and independent of the concentration of substrate up to a
certain point. Then it follows the first or higher order reactions. The kinetic curve has a shape
of a line.
v = k[A]
0
= k

The Michaelis-Menten plot:
The Michaelis-Menten plot illustrates mathematically the relationship between the initial
velocity v
0
and the substrate concentration [S].
v
0
= (v
max
[S]) / (K
M
+ [S])
The Michaelis constant K
M
is the substrate concentration at which v
0
is half the maximal
velocity at a certain substrate concentration. It states the affinity of an enzyme for a substrate.
K
M
does not vary with the concentration of enzyme.
[S] << K
M
: The enzyme has low affinity for the substrate because a high concentration of
substrate is needed to half-saturate the enzyme. The velocity of the reaction is approximately
equal to the substrate concentration. The reaction is said to be of first order.
v
0
= (v
max
[S]) / K
M
= k[S]
1


[S] >> K
M
: The enzyme has high affinity for the substrate because a low concentration of the
substrate is needed to half-saturate the enzyme. The velocity is constant and equal to v
max
. The
rate of reaction is independent on the substrate concentration and the reaction is of zero
order.
v
0
= (v
max
[S]) / [S] = v
max
= k[S]
0


4. The enzyme activity (the term, units of enzyme activity U and katals,
catalytic concentration) and assays of enzymes (the conditions used in
enzyme assays, the kinetics arranged by the substrate concentration,
the kinetic and/or constant-time method):

8


Enzyme activity:

Enzyme kinetics is the study of chemical reactions catalyzed by enzymes. By studying its
reaction rate and effects of varying conditions and inhibitors its function in human
metabolism can be determined.
Enzyme activity is the number of moles of a substrate that is converted by an enzyme by unit
time. The SI unit for enzyme activity is katal (kat, mol/s) and is the same thing as catalytic
activity. However, katal is a rather large unit and U is more frequently used (mol/s).
Catalytic concentration is the catalytic activity of the enzyme in one litre of solution (kat/l
or mol/ls).

Assays of enzymes:

Assays of enzymes measure either the consumption of substrate or production of product over
a certain time and serve to estimate the reaction velocity and thus also the activity of the
enzyme. The assumption that the reaction rate is proportional to the amount of enzyme is
made and there is an excess of substrate over enzyme Because of this the initial reaction rate
is proportional to the amount of enzyme present in the sample. There are a number of methods
used for this purpose:

Kinetic method: Changes in substrate or product concentrations are measured during the
reaction by means of a spectrophotometer. The absorbance of the sample is proportional to the
concentration of products formed. A kinetic curve needed and the initial velocity is measured.
The evaluation of this method is very exact.
A = l c
The initial reaction velocity v
0
is determined from this.
Constant time method: The average reaction velocity is calculated. Reactions proceed for a
fixed time, and then stopped by inactivation of enzyme. The concentration of product is then
determined and the reaction velocity is then calculated from this. This method is less exact
than the kinetic method.

5. Factors affecting catalytic activity of enzymes (the optimal conditions,
activators and inhibitors, basal types of inhibitors, the distinguishing
competitive from noncompetitive inhibition using saturation curves.
The roles of metal ions in enzymatic catalysis (cofactors, metals as
activators and inhibitors, examples of metalloenzymes):

Factors affecting the catalytic activity of enzymes:

- Salt concentration: Most enzymes cannot tolerate high salt concentrations since the
ions interfere with weak ionic interactions of the proteins.

- Temperature: Too high temperature might lead to denaturation of the protein since

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the forces between the bonds get instable and break. Optimal is slightly higher kinetic
energy of the molecules that will enable the substrate to bind easier to the enzyme and
they will overcome the activation energy barrier easier. Enzymes have optimal
function between 35-40
o
C.

- pH: Enzymes have different optimum pH values that will make them have as many
ionized groups as possible. Generally enzymes have their optimal pH at physiologic
pH 7,4, but there are exceptions like e.g. pepsin that resides in the stomach and that
have optimum pH at 2,0. If the pH is too low or too high the three-dimensional
structure of the enzyme might be destroyed due to ionic- and hydrogen bonds that
might break.

- Substrate saturation: If the substrate concentration is in excess to the enzyme, the
enzyme will be saturated and the reaction velocity will be proportional to the enzyme
concentration. When all the active sites of the enzyme are occupied it doesnt matter
how much substrate we add, the reaction velocity will only depend on the
concentration of the enzyme.

- Level of crowding: In living systems other macromolecules that dont participate in
the reaction are present. These serve to create a barrier for the access of enzyme to
substrate and this slows down the reaction rate. This phenomenon is called
macromolecular crowding. This explains why a reaction occurring in vitro can differ
radically to the same reaction in vivo.

Activators and inhibitors:

Enzyme activators are molecules that bind to enzymes and increase their activity. These
molecules are often involved in allosteric regulation of enzymes in metabolism. Effector
molecules that increase the enzymes catalyzing rate are referred to as allosteric activators
that bind to the enzyme in the allosteric site that is a distinct site from the active site.
Molecules that decrease the enzymes activity are called allosteric inhibitors. Allosteric
activators can e.g. give feed-forward in a reaction from upstream substrates. Generally, when
the enzyme binds to an allosteric effector a conformational change takes place of the enzyme
that affects the active site. The saturation curve of allosteric effectors is sigmoidal.
Allosteric inhibitors are molecules that by their binding to the allosteric site of the enzyme
reduce the activity of the enzyme and act as negative allosteric effectors. Two type of
allosteric inhibition are distinguished; K-series and V-series enzymes. In K-series allosteric
enzymes the K
M
value is raised but the v
max
remains unaffected. In V-series allosteric
inhibitors lower v
max
without affecting K
M
.

Inhibitors:

Kinetically, two types of inhibitors are distinguished based on if raising the substrate
concentration does or does not overcome the inhibition.
The effects of competitive inhibitors can be overcome by raising the substrate concentration.

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Most frequently the competitive inhibitor inhibits by binding to the substrate-binding place of
the active site. Therefore, the structure of competitive inhibitors tends to be very similar to the
substrate and is thus referred to as substrate analogues. An example of a substrate analogue
is malonate that competes with succinate to bind to succinate dehydrogenase. Succinate is
converted to fumarate, but this is blocked when malonate binds. A competitive inhibitor acts
by decreasing the number of available active sites of the enzyme, and thus the production of
products. The concentration of substrate that has to be added to decrease the activity of the
inhibitor depends on the concentration of the inhibitor present, its affinity for the enzyme and
the affinity of the enzyme to its substrate. A competitive inhibitor has no effect on v
max
, but it
raises the apparent K
M
for the substrate. The lower the K
i
(affinity of enzyme for inhibitor) the
more efficient is the inhibitor.
In non-competitive inhibition, binding of the inhibitor does not affect the binding of the
substrate and there can be both formations of EI and EIS complexes. The saturation curve
where non-competitive inhibitors are involved therefore exhibit a curve where the K
M
of the
enzyme and substrate is unaffected, but where v
max
is lowered. Non-competitive inhibitors
bind to another site than the substrate and therefore they exhibit no similarities to the
substrate. An example of non-competitive inhibition is the inhibition of synthesis of
cholesterol by statins that inhibit HMG-CoA reductase. In simple non-competitive
inhibition the E and EI complexes have identical affinity for the substrate but the EIS
complexes convert the product at a very low rate.
Complex non-competitive inhibition occurs when binding of the inhibitor affects the affinity
of the enzyme to the substrate and therefore the limit between competitive and non-
competitive inhibition can be diffuse for some inhibitors.
Irreversible inhibitors are usually compounds of non-biological origin that bind covalently
to the enzyme which makes it impossible for the enzyme to bind a substrate. Heavy metals
bind irreversibly to enzymes during isolation. Irreversible inhibitors chemically modify the
enzyme when binding to it and it can only be removed by dialysis of the solution. Suicide
inhibitors are a part of this group that are recognized by the enzyme as substrates but when
they are catalyzed they form active intermediates that inhibit the enzyme, e.g. Aspirin.
Reversible inhibitors bind to the enzyme in a loose manner with non-covalent bonds and it
can easily dissociate from the EI complex. It is further divided into non-competitive and
competitive inhibition depending on if raised substrate concentration can eliminate the
activity of the inhibitor.

Metal ions in enzymatic catalysis:

Metalloenzymes have a metal ion as a prosthetic group that usually is located inside a pocket
of the enzyme and they include most trace elements in the human body. Enzymes that have
metals as cofactors are called metal-activated enzymes. Examples of metalloenzymes are
vitamin B12-dependent enzymes like methionine synthase that catalyzes the conversion
from homocystein to methionine. Vitamin B12 contains the metal Co (cobalt) that mediates to
lower the activation energy and thus facilitating the reaction to take place. Another example
of metalloenzymes is carbonic anhydrase that has zinc incorporated into its structure.
Another example is calcium that is required in the human diet since it is required for the
activity of many enzymes where it serves as allosteric regulator. Some of these enzymes that
it regulates is e.g. nitric oxide synthase and protein phosphatises which whom it forms

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complexes with calmodulin. Due to this it is not generally only referred to as a cofactor of the
enzymes it regulates, but more as a cell signalling molecule.

7. Regulation of the catalytic activity of enzymes by covalent modification
(namely conversions of proenzymes, reversible phosphorylations, activation
of proteinkinases). Allosteric proteins and enzymes (cooperativity, allosteric
activation and inhibition):

Regulations of the catalytic activity of enzymes:

Proenzymes are inactive forms enzymes that are synthesized as inactive proproteins.
Selective proteolysis converts the proenzymes into active enzymes by one or several clips.
Examples of protein secreted as immature proenzymes are insulin (proinsulin), the digestive
enzymes pepsin (pepsinogen), trypsin (trypsinogen), and chymotrypsin
(chymotrypsinogen).
Degradation proteins, proteases, are also digested as proenzymes. This is especially
important for protection of the tissue where the enzyme originates from since it will be
prevented from autodigestion.
Selective proteolysis involves one or more specific proteolytic clips that may or may not
result in the separation of the resulting peptides. Most importantly, these clips result in the
activation of the enzyme and the creation of a catalytic site.
Among covalent modifications that regulate protein activity (e.g. methylation, acetylation),
phosphorylation and dephosphorylation is the most common way of regulation. Protein
kinases phosphorylate proteins by catalyzing transfer of the terminal phosphoryl group of
ATP to the hydroxyl groups of seryl, threoyl, or tyrosyl residues forming O-phosphoseroyl,
O-phosphotheroyl, and O-phosphotyroyl residues. Some protein kinases target the side
chains of histidyl, lysyl, arginyl, or aspartyl groups. The unmodified state of the protein can
be regenerated by hydrolytic cleavage of the phosphoryl groups catalyzed by protein
phosphatises. By the phosphorylation/dephosphorylation of proteins the catalytic activity of
the enzymes can be regulated very precisely and the enzymes are only active for as long as
they have a function. A reason for that phosphorylation is so widespread is that the amino
acids the phosphoryl group binds to lie relatively far from the catalytic site of the enzyme
which is beneficial for its catalytic activity. For some enzymes phosphorylation means
conversion to an active form, while for others it means deactivation of the enzyme. Examples
of enzymes regulated by phosphorylation is glycogen synthase, pyruvate dehydrogenase
and citrate lyase.
Protein kinases and protein phosphatases usually act on more than one protein and are
themselves interconverted between active and inactive form by the binding of second
messengers or by covalent modifications by phosphorylation/dephosphorylation. The fact that
they act on more than one enzyme means an ability to control several metabolic processes at
once.

Allosteric enzymes and proteins:

See question 6.

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Cooperative binding occurs in enzymes with several allosteric sites where the binding of one
molecule increases the affinity of binding more molecules. Cooperativity results from the
interaction between the binding sites where bonds are broken as one molecule is bonded and
therefore it gets easier as more and more bonds are broken. Positive and negative
cooperativity can be distinguished depending on if the affinity increases or decreases.
Allosteric enzymes exhibit a sigmoidal dependence on the substrate concentration (saturation
curve). It can be compared to the binding of oxygen to deoxyhemoglobin.






8. The roles of hydrogen and oxygen in the energy exchange of living
systems (foodstuffs for chemotrophs, three stages in the extraction of
energy from nutrients, reducing equivalents), production of ATP by
oxidative phosphorylation and by phosphorylation on the substrate level:
Chemotrophs:

Chemotrophs are organisms that obtain energy from oxidation of electron donors in their
environment obtained from chemical energy. Chemotrophs can be divided into chemotrophic
autotrophs and chemotrophic heterotrophs. Chemotrophic autotrophs (chemoautotrophs)
can in addition to deriving energy from chemical reactions, synthesize all necessary organic
compounds from carbon dioxide. Bacteria and green leaf plants are included in this group.
Chemotrophic heterotrophs (chemoheterotrophs) are unable to fix carbon to synthesize
necessary organic compounds. Heterotrophic organisms obtain free energy by coupling their
metabolism to the breakdown of complex organic molecules in their environment. In all these
organisms, ATP plays a central role in the transference of energy from exergonic processes to
endergonic processes. Most microorganisms and higher animals are included in this group.

3 stages in extraction of energy from nutrients:

Nutrients in food (lipids, saccharides, and partially proteins) contain carbons with low
oxidation number that are continuously degraded (oxidized) to various intermediates that
release CO
2
in decarboxylation reactions. Electrons and H atoms are transferred to redox
cofactors (NADH and FADH
2
) and transported to the terminal station, the respiratory chain.
The oxidation of the reduced cofactors serves to produce ATP. Before combustion a
conversion into acetyl-Co A takes place that enters the citric acid cycle, glucose enters first
the glycolysis.
1. Hydrolysis of biopolymers to smaller units in the digestive tract (no net yield of
energy)
2. Metabolism of glucose acetyl-CoA + small amount of ATP + reduced cofactors
(NADH + H
+
)
Metabolism of amino acids pyruvate, acetyl-CoA, intermediates of CAC +

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some reduced cofactors.
Metabolism of fatty acids -oxidation of FA Acetyl-CoA + reduced
cofactors
3. Oxidation of acetyl-CoA in the CAC ATP + reduced cofactors
Oxidation of reduced cofactors in the RC ATP (highest yield)

Production of ATP:

The production of ATP is vital for three major purposes in living systems:
1. Mechanical work
2. Active transport of molecules and ions
3. Synthesis of macromolecules and other biomolecules
ATP can be formed both by oxidative phosphorylation in the respiratory chain (95%) and by
substrate-level phosphorylation (5%). Substrate-level phosphorylation occurs e.g. in the
citric acid cycle and the glycolysis. When high-energy compounds are cleaved energy is
released that is used for synthesis of compounds that require input of energy to be formed.
This phenomenon is called coupling and involves exergonic processes (spontaneous) that are
coupled to endergonic processes (non-spontaneous).
G = H - ST
When Gibbs free energy has a negative value the reaction is said to be spontaneous and
exergonic. In exergonic reactions the product has less energy than the reactants and the
remaining energy difference is released as free energy. Endergonic reaction can only occur if
they receive energy, therefore they are coupled with exergonic reactions that release energy.
Their products have a higher value of internal energy in comparison to the reactants.
Therefore, energy have to be added for this reaction to occur. In this way ATP can be formed
by the release of free energy from exergonic processes of macroergic compounds inorganic
phosphate can be added to ADP. In other reactions mostly ATP is used:
Glucose + P
i
Glucose-6-P + H
2
O
ATP + H
2
O ADP + P
i


Oxidative phosphorylation occurs in the respiratory chain and is based on an electrochemical
potential gradient between the matrix and intermembranespace across the inner
mitochondrial membrane. Protons are pumped into the inner mitochondrial membrane and
according to the principle of osmosis, concentration differences are to be equalized. Inorganic
phosphate is joined to ADP in by the ATPsynthase as the potential differences are allowed to
be equalized.
Phosphagens are high-energy compounds that serve as high-energy phosphate compounds
that can be found in muscle tissue. It serves as a phosphate pool for rapid ATP synthesis that
is more immediate than glycolysis which is important in unexpected situations. Phosphagens
include e.g. creatine, creatine phosphate and phosphocreatine. When the ratio ATP/ADP is
high their concentration increases.

9. Pyridine nucleotide-dependent dehydrogenases (structures of

14

coenzymes, function):

Pyridine nucleotide-dependent dehydrogenases:

Dehydrogenases are linked to coenzymes because they need a hydrogen acceptor. Important
coenzymes in the body are NADH, NADPH, FMN and FADH
2
. Dehydrogenases perform two
major functions:

1. Transfer of hydrogen from one substrate to another in a coupled oxidation-
reduction reaction: These dehydrogenases are specific for their substrate but often
utilize common coenzymes. These reactions are reversible. They are very useful since
they can occur without the presence of oxygen and can thus function under anaerobic
conditions because one substrate is oxidized on the expense of another substrate.

2. As components in the respiratory chain of electron transport from substrate to
oxygen.
NADH: is a dinucleotide formed from the vitamin niacin or from simple building blocks
from tryptophan or aspartic acid. It is involved in oxidation-reduction reactions where it
accepts or donates hydrogen. It has therefore an oxidized form NAD
+
and a reduced form
NADH + H
+
(the molecule can only carry one H atom).
NADPH: is a dinucleotide involving phosphate as well that is also formed in the body from
the vitamin niacin. The reduced form is NADPH and the oxidized form is NADP
+
. It
participates in anabolic reactions such as lipid and nucleotide synthesis.

Generally, NAD-linked dehydrogenases catalyze oxidoreduction reactions in the oxidative
part of the metabolism, especially in glycolysis, citric acid cycle and respiratory chain.
NADP-linked dehydrogenases are generally found in reductive syntheses, as in
extramitochondrial fatty acid synthesis, steroid synthesis and also in the pentose phosphate
pathway.

10. Flavoproteins (structures and function of the flavin prosthetic group,
function of flavin dehydrogenases):

Flavopriteins:
Flavoprotein enzymes include FAD or FMN as prosthetic groups. Oxidation and reduction is
not as simple as for NAD and NADP, but consists of a two-step reaction where 2 H atoms are
added one at a time in two steps. Metalloflavins contain one or more metals as essential
cofactors. Flavoproteins are important components of complexes I and II where they can
transfer two electrons or accept one electron to form semiquinone. Flavoproteins belongs to
the oxidases out of all oxidoreductases.
FADH
2
: Consists of a riboflavin (vitamin B
2
) linked by phosphate to an adenosine
nucleotide. It exists in an oxidized state FAD
+
and a reduced state FADH
2
. Flavin-dependent
dehydrogenases are concerned e.g. with electron transport in the respiratory chain. Note that
FAD is an aromatic system while FADH
2
is not. Another use of FADH
2
is in the -oxidation
where FAD serves as a coenzyme to the acyl CoA dehydrogenase.

15

FMN: It is also formed by the vitamin riboflavin and is a prosthetic group of many
oxidoreductases. It is a stronger oxidizing agent than NAD.

11. Cytochromes of the mitochondrial respiratory chain (the main
structural features, the roles in mitochondrial complexes) and of
monooxygenase hydroxylating systems (cytochrome P450):

Cytochromes:

Cytochromes are iron-containing hemoproteins in which the iron atom oscillates between
Fe
3+
and Fe
2+
during oxidation and reduction. Except for cytochrome oxidase (terminal
component of the respiratory chain) all cytochromes are classified as dehydrogenases. They
are involved as carriers of electrons (1 electron each) from flavoproteins to cytochrome
oxidase. Identifiable cytochromes in the respiratory chain are: cytochrome b, c
1
, c, and
cytochrome oxidase. Cytochromes are not only found in the respiratory chain but also in the
endoplasmatic reticulum where cytochrome b
1
and cytochromes P450 are found.
Cytochrome oxidase (aa
3
) is a hemoprotein found as the terminal carrier component of the
respiratory chain in mitochondria. It has two heme groups each with an iron oscillating
between Fe
2+
and Fe
3+
and also two atoms of Cu each associated to the heme groups. It
transfers electrons resulting from the oxidation of substrate molecules by dehydrogenases to
their final acceptor, oxygen. It is inhibited by cyanide, carbon monoxide, and hydrogen
sulphide.
Cytochrome c is a bit special since it is water-soluble and peripheral protein complex that
moves on the outer side of the inner mitochondrial membrane.
Cytochromes P450 are a group of heme-containing monooxygenases (incorporate only one
atom of molecular oxygen into the substrate, the other oxygen atom is incorporated into
water). The enzymes are located in the endoplasmatic reticulum of the liver and intestine, but
also in mitochondria of some tissues. Cytochromes P450 are found together with cytochrome
b
1
and they are important for detoxification. The rate of detoxification by P450 determines
how long a medicinal drug will be active.
Mitochondrial cytochrome P450 are found in steroidogenic tissues like the adrenal cortex,
testis, ovary and placenta and are involved in the synthesis of steroids from cholesterol.

12. Mitochondria (general structure, overview of the main roles in
metabolism). Transporter systems in the inner mitochondrial membrane
(transport and transporter types, examples):

Mitochondria:

The mitochondria is a cellular organelle about 10 m in diameter and is composed of an outer
and inner membrane, a intermembrane space between these membranes and a matrix. The
inner membrane forms crista that are folding that increases the surface area of the inner
membrane which is important for the maximal production of ATP since more space is
available for the complexes of the respiratory chain to occur. Major pathways occurring in the
mitochondria are:

16

1. Respiratory chain: One of the major tasks in metabolism is to produce ATP by
oxidative phosphorylation.
2. Citric acid cycle: Pyruvate is transported through active transport into the matrix
where the citric acid cycle takes place. It is oxidized and combines with CoA forming
in total acetyl-CoA, CO
2
and one NADH. Then it joins oxaloacetate and the citric acid
cycle is initiated.
3. -oxidation: Fatty acids are transported into the mitochondria via carnitine and
oxidized to acetyl-CoA in the matrix. The yield of every cycle is 14 ATP.
4. Steroid synthesis: The synthesis starts and ends with dimethylallylpyrophosphate
(DMAPP) and isopentenyl pyrophosphate (IPP) with acetyl-CoA as building blocks.

The inner mitochondrial membrane is more impermeable than the outer membrane. It is richer
in cardiolipin and it is impermeable to charged molecules and other molecules. This is
important in the establishment of a proton gradient across the inner membrane, but it causes
problems with transport of certain molecules, and thus several transport systems have been
developed. Molecules permeable to the inner membrane are O
2
, CO
2
, H
2
O, and NH
3
. See
attached paper for further transport systems.
- Aspartate/Malate shuttle: The malate shuttle is believed to be the universal transport
system in mitochondria for the transport of NADH from the cytosol to the matrix of
the mitochondria. This is due to the impermeability of the inner mitochondrial
membrane to NADH and thus malate carries the reducing equivalents across the
membrane. It consists of four components; malate dehydrogenase (in the
mitochondrial matrix and intermembrane space), aspartate aminotransferases (in the
mitochondrial intermembrane space and matrix), malate--ketoglutarate antiporter
(in the inner membrane), glutamate-aspartate antiporter (in the inner membrane).
The primary enzyme of the malate shuttle is the malate dehydrogenase that is present
both as cytosolic and mitochondrial that catalyze in opposite directions. First in the
cytosol, malate dehydrogenase reacts with oxaloacetate and NADH to form malate
and NAD
+
. Here 2 H atoms are attached to oxaloacetate in the form of malate. Once
malate is formed it is transported from the cytosol to the mitochondrial matrix by
malate--ketoglutarate antiporter simultaneously as -ketoglutarate is transported from
the matrix to the cytosol by the same antiporter. As malate has reached the matrix it is
converted back into oxaloacetate and NADH (1 H
+
is released) by malate
dehydrogenase.
Oxaloacetate is then transformed into aspartate by mitochondrial aspartate
aminotransferases due to that oxaloacetate is impermeable to the inner membrane.
Since aspartate is an amino acid an amino acid radical need to be added to
oxaloacetate which is provided by glutamate that is then transformed into -
ketoglutarate by the same enzyme. The second antiporter, glutamate-aspartate
antiporter, transports the aspartate from the matrix to the cytosol and cytosolic
glutamate to the matrix. Once aspartate is in the cytosol it is converted by cytosolic
aspartate aminotransferases to oxaloacetate. The NADH that then joins oxaloacetate
comes from the glycolysis and the oxidized NAD
+
can then be used for another round
of glycolysis. The two H
+
are shipped to NAD
+
that is oxidized in the respiratory chain
and that can be reduced again and ready for another round.


17

- Glycerophosphate shuttle: This shuttle occurs only in brain and white muscle, but
deficient in heart muscle. The glycerophosphate shuttle also serves to transport
reduced NADH + H
+
from the glycolysis to the respiratory chain where it is to be
oxidized. In this reaction cytoplasmic glycerol-3-phosphate dehydrogenase converts
dihydroxyacetone phosphate to glycerol-3-phosphate by oxidizing one molecule of
NADH + H
+
to NAD
+
. Glycerol-3-phosphate gets converted back to dihydroxyacetone
phosphate by a membrane-bound glycerophosphate dehydrogenase, this time
reducing one molecule of FAD to FADH
2
. FADH
2
then reduces coenzyme Q
(uniquinone to ubiquinol) to QH
2
which enters into oxidative phosphorylation. This
reaction is irreversible. The FADH
2
goes to the respiratory chain.

- Carnitine transport system: Free fatty acids that have first been activated by acyl-
CoA are transported to the intermembrane space by carnitine palmitoyltransferase I to
form acylcarnitine which is able to penetrate the inner mitochondrial membrane.

- Ionophores: Lipophilic molecules that facilitate the transport of charged ions across
biological membranes and that occur in the inner mitochondrial membrane due to its
impermeability to charged molecules and ions.


13. The origin of reactive oxygen species, oxygen radicals detoxification
(enzymes and natural antioxidants):

Reactive oxygen species (ROS):

Reactive oxygen species contain oxygen with an unpaired valance electron which makes them
highly reactive. They are formed in the body during metabolism of oxygen and are important
in cell signalling. During exposure to heat and UV-radiation the amount of reactive oxygen
species increase that lead to damages of cells and that are suspected to influence the aging
process and the formation of cancer cells. There are some main forms of reactive oxygen
species in the body:
Radicals: Anion/Neutral/Cation:
Superoxide O
2
-

Hydroxyl radical OH
Peroxyl radical* ROO
Alkoxyl radical RO
Hydroperoxyl radical HOO
Nitric oxide NO
Hydrogen peroxide H-O-O-H


Hydroperoxide* R-O-O-H
Hypochlorous acid HClO
Singlet oxygen
1
O
2

Peroxynitrite ONOO
-


18












See attached paper for individual reactions.
Reactive oxygen species are intermediates of oxygenase and oxidase reactions and are
detoxified by P450. Reactive oxygen species have good bactericidal effects and they are also
important in cell signalising. Reactive oxygen species can give damages to PUFA:s, proteins
and DNA. PUFA:s give rise to peroxides and aldehydes and destroy lipid membranes which
in turn destroys its function with permeability etc. Proteins might aggregate and there will be
changes in enzyme activity. DNA might be damaged so that mutations arise and there will be
errors in translation and synthesis of proteins.
Antioxidants:
An antioxidant is a molecule capable of slowing down or preventing the oxidation of other
molecules. Oxidations can produce free radicals that can initiate chain reactions and thus
cause damages to the cell. Antioxidants react with these reactive intermediates and prevent
these chain reactions from taking place by being oxidized themselves. Antioxidants therefore
serve as reducing agents.
Antioxidants can exist in the form of enzymes like e.g. superoxide dismutase, catalase, and
glutathione peroxide. There are also low-molecular antioxidants that are reducing
components of compounds like e.g. phenolic OH (tocopherol, flavenoids), enolic OH
(ascorbate), and SH (glutathione, dihydrolipoate).
See attached paper for elimination reactions.
We can also distinguish Lipophilic and hydrophilic antioxidants. Major Lipophilic
antioxidants are tocopherols (plant oils, nuts, seeds), cartenoids (fruits, vegetables), and
ubiquinol (formed in the body from tyrosine). Major hydrophilic antioxidants are L-
ascorbate (fruits, vegetables, potatoes), flavenoids (fruits, vegetables, tea, wine),
dihydrolipoate (made in the body from cysteine), uric acid (catabolite of purine bases), and
glutathione (made in the body from cysteine).

14. The mitochondrial respiratory chain (function, the main components of
the mitochondrial complexes, the proton-motive force, the respiratory
control):
Nitronium NO
2
+


19


The mitochondrial respiratory chain:

Enzymes of the citric acid cycle and -oxidation are contained in the matrix along with
enzymes of the respiratory chain. The respiratory chain collects and transports reducing
equivalents, directing them to their final reaction with oxygen to form water. Reducing
equivalents refer to chemical species that transfer the equivalent of one electron in redox
reactions. Finally, the conversion of oxygen into water activates the machinery for oxidative
phosphorylation, the process where free energy is trapped as high-energy phosphate.

The electron transport chain couples a chemical reaction between an electron donor (such as
NADH) and an electron acceptor (such as oxygen) to the transfer of H
+
ions across a
membrane, through a set of mediating biochemical reactions. The H
+
are pumped to the
intermembrane space that creates an electrochemical gradient across the inner membrane that
is used in the production of ATP. The respiratory chain is a major site for electron leakage to
oxygen that produces oxygen radicals that is responsible for oxidative stress.

The electron transport chain consists of a spatially separated series of redox reactions in
which electrons are transferred from a donor molecule to an acceptor molecule. The
underlying driving force of these reactions is based on the principle of Gibbs free energy.
Substrates in the upper part of the reaction chain have a more negative value of Gibbs free
energy and are more exergonic and proceed more spontaneously. Their value of electric
potential is also more negative implying that these substance are easily oxidized, while
substances lower in the reaction chain have a less negative value and are not as easily
oxidized.
The respiratory chain has four complexes; complex I (NADH-Q-oxidoreductase), complex
II (succinate-Q-reductase), complex III (Q-cytochrome c-reductase), and complex IV
(cytochrome c-oxidase):
- Complex I: In complex II two electrons are removed from NADH and transferred to
uniquinone Q that is then reduced to QH
2
. This event is accompanied with the
translocation of four H
+
to the intermembrane space. Complex I is the main site for
premature electron leakage that may give rise to superoxide.
The pathway of electrons occurs as follows:
NADH is oxidized and its electrons are transferred to FMN that is by this reduced to
FMNH
2
(in a two-electron step, semiquinone intermediate). Each electron is then
transferred to a Fe-S cluster, from the Fe-S cluster to ubiquinone that is then reduced
to ubiquinol. The transfer of the first electron to ubiquinone forms semiquinone and
finally the transfer of the second electron forms ubiquinone. Four protons are pumped
during this action.

- Complex II: In complex II, FADH
2
is formed when succinate is converted to
fumarate in the citric acid cycle and its two electrons are transferred to ubiquinone that
is reduced to ubiquinol via several Fe-S centres. Other electron donors (e.g. glycerol-
3-phosphate, fatty acids) also enter their electrons here via FAD.
Complex II consists of four subunits; SDHA, SDHB, SDHC, and SDHD. Complex II
does not participate in the pumping of electrons.


20

- Complex III: Electrons are passed from QH
2
via complex III to cytochrome c. This
process is called the Q cycle and involves cytochromes c
1
, b
L
, b
H
, and a Rieske Fe-S
(one of the Fe atoms is linked to two histidine-SH groups rather than two cysteine-SH
groups. One round of the cycle involves the oxidation of QH
2
to Q, releasing 4 H
+
into
the intermembrane space, and the reduction of Q to QH
2
, causing 2 H
+
to be taken up
from the matrix. Note that while Q carries two electrons, cytochromes carry only one,
thus the oxidation of one QH
2
is coupled to the reduction of two molecules of
cytochrome c via the Q cycle. Complex III may leak electrons to produce superoxide.

- Complex IV: In complex IV four electrons are removed from four molecules of
cytochrome c and transferred to oxygen. Reduced cytochrome c is oxidized by
complex IV with the concomitant reduction of O
2
to two molecules of water. This
transfer of four electrons from cytochrome c to O
2
involves two heme groups (a and
a
3
), and Cu. Electrons are passed initially to a Cu centre, which contains two Cu
atoms linked to two protein cysteine-SH groups (resembling Fe-S), then in sequence to
heme a, heme a
3
, and then to a second Cu centre, Cu
B
, which is linked to heme a
3
, and
finally to O
2
. Of the eight H
+
removed from matrix, four are used to form water and
the other four are pumped to the intermembrane space. O
2
is tightly bound to complex
IV until it is fully reduced, this to minimize the origin of superoxide and peroxide.

The proton motive force:

The proton motive force is another name for the chemiosmotic potential that drives the
oxidative phosphorylation, production of ATP. It involves two components, a difference in
pH and potential between the matrix and the intermembrane space.

pH : G = RT ln ([H
+
]
out
/[H
+
]
in
) = 2.3 RT(pH
out
pH
in
)

+: G = nF+
The difference in electric potential depends not only on the proton concentration, but also on
concentrations of other ions. The proton motive force is expressed in potentials (mV/mol H
+

transferred):
p = G / nF = + + 60 pH


The respiratory control:

The rate of respiration of mitochondria is controlled by the availability of ADP. This is
because phosphorylation and oxidation are tightly coupled. Under certain conditions, the
availability of inorganic phosphate might regulate the respiration. The ATP/ADP transporter
that transports cytosolic ADP into the matrix might also be rate-limiting. Of course, the
availability of oxygen is also one crucial rate-limiting factor. Also, the capacity of the
respiratory chain itself can be rate-limiting when all the substrates and components are
saturated.


21

15. Ubiquinone (structure, function) and iron-sulphur proteins (the term,
function):

Ubiquinone:

Ubiquinone or coenzyme Q is a fat-soluble substance present in most eukaryotic cells that is a
component of the respiratory chain, responsible for 95% of the ATP production in the body. It
is ingested in the diet and meat and fish are the best sources. The benzoquinone portion of
ubiquinone is synthesized from tyrosine, whereas the isoprenoid chain is derived from acetyl-
Co A from the mevalonate pathway. Statins that reduce cholesterol synthesis and which in
turn also regulated the production of steroids will decrease the amount of acetyl-CoA that can
be used in ubiquinone synthesis. UV-radiation and aging also decreases the amount of
ubiquinone formed in the body.
Its function is to accept electrons from complex I and II and transfer these to complex III. It
accepts 2 electrons from NADH from complex I that reduces it to QH
2
. It also receives 2
electrons from FADH
2
from complex II that reduces another ubiquinone molecule to
ubiquinol. In total it receives 4 electrons that are pumped to complex III. Ubiquinone has
three intermediate states; oxidized ubiquinone, intermediate semiquinone, and reduced
ubiquinol.

Iron-sulphur proteins:

iron-sulphur proteins are non-heme proteins found in complex I, II, and III. They may contain
one, two or four Fe atoms linked to inorganic sulphur atoms or via cysteine-SH groups to the
protein. Fe-S proteins take part in single electron transfer in whom one Fe atom undergoes
oxidoreduction between Fe
2+
and Fe
3+
. Fe-S proteins are present in complexes I and II.

16. Energetics of the respiratory chain, oxidative phosphorylation
(structure and function of the ATP synthase, coupling of phosphorylation to
electron transport, respiratory control, uncouplers):

Energetics of the respiratory chain:

Transfer of 2 electrons from NADH to O
2
provides 3 ATP
Transfer of 2 electrons from FADH
2
to O
2
provides 2 ATP


Oxidative phosphorylation:

The proton motive force drives a membrane-located ATP synthase that in the presence of
ADP and P
i
forms ATP. ATP synthase is embedded in the inner mitochondrial membrane, the
same location of the complexes of the respiratory chain. The head of the ATP synthase, the
F
1
segment projects into the matrix and contains the phosphorylation mechanism and 5
subunit types; o
3
,

|
3
, , o, c. F
1
is attached to a rotating F
0
unit that spans the inner
mitochondrial membrane and forms a proton channel by its several c units. Between F
1
and

22

F
0
is a connecting segment. The a, b
2
, and o units attaches the F
1
to the inner membrane and
makes sure it is not rotating.
The current model of the mechanism of ATP synthase (alternating catalytic model) states
that the proton motive force drives protons through the proton channel of F
0
where the c units
rotate as protons flow through it. The c units are attached to the gamma unit of the F
1

segment, only the gamma segment rotates in this segment. The rotating gamma unit causes
conformational changes in the three catalytic nucleotide binding sites of the unit leading to
the joining of ADP and P
i
to ATP. There are three states; open, loose, and tight. In the open
state ADP and P
i
get access to the active site. In the loose state the enzyme closes up around
the molecules and binds loosely to them. In the tight state the enzyme undergoes
conformational changes and forces the ADP and P
i
to join together. The enzyme then returns
to the open state again, ATP is released and new ADP and phosphate enter the active site.

See question 14 for respiratory control.

Uncouplers:

An uncoupling protein is an inner mitochondrial membrane protein that can dissipate the
proton gradient before it can be used to provide energy for oxidative phosphorylation. There
are 5 main types known in mammals; UCP1, UCP2, UCP3, UCP4, and UCP5. UCP1 is also
known as thermogenin that is important in hibernating animals where it generates heat but
displacing the proton gradient, the release of H
+
causes the release of free energy that is
converted into heat. Other uncouplers are ethanol and salicylic acid that will generate heat is
taken in excess. Another uncoupler is 2,4-dinitrophenol. In a way, uncouplers can be said to
be proton Ionophores.

17. Peroxidation of lipids. Tocoferols and futher lipophilic antioxidants:

Peroxidation of lipids:

Peroxidation (auto-oxidation) of lipids exposed to oxygen is responsible not only for the
deterioration (=frsmring) of foods (rancidity = hrskning) but also for damage to tissues in
vivo, where it might be the cause of cancer, inflammatory deseases, aging and arterosclerosis.
Free radicals formed during lipid peroxidation is ROO, RO, and OH. They are produced
during peroxide formation from fatty acids containing methylene-interupted bonds, i.e.
those found naturally in polyunsaturated fatty acids (PUFA), double bond conaining fatty
acids. Other radicals steal electrons from the lipids, especially the methylene group lying
between the double bonds is exposed of this risk since it contains reactive hydrogens. Lipid
peroxidation is a chain reaction providing continous supply of free radicals that initiate further
peroxidation and thus has potentially devastating effects. Lipid peroxidation has three main
stages:
1. Initiation:
ROOH + Metal
(n)+
ROO + Metal
(n-1)+
+ H
+



23

X + RH R + XH

2. Propagation:
R + O
2
ROO

ROO + RH ROOH + R etc.

3. Termination:
ROO + ROO ROOR + O
2

ROO + R ROOR

R + R RR

To control the lipid peroxidation antioxidants are used. Many antioxidants are used as food
additives to improve the health of the population, and many antioxidants are present in fruit
and vegetables as vitamins. Examples of naturally occuring antioxidants are vitamin E
(tocopherol), urate, vitamin C (ascorbate), beta-carotene at low pO
2
. Antioxidants fall into
two classes:
- Preventive antioxidants: Reduces the rate of chain reactions. Examples are catalase,
superoxide dismutase, glutathione, selenium, chelators of metal ions like EDTA,
and DTPA.
- Chain-breaking antioxidants: Antioxidants that interfere with the chain propagation.

Lipid peroxidation is catalyzed by heme compounds, and lipoxygenases found in platelets
and leukocytes. Other products than free radicals include oxysterols from cholesterol, and
isoprostanes formed from PUFA:s such as arachidonic acid.

Tocopherol:

Tocopherol is a class of compounds which have vitamin E activity. It is a common food
additive and a lipophilic antioxidant in lipid peroxidation. Main food sources are plant oils,
nuts, seeds, and germs. It reduces peroxyl radicals of phospholipids to hydroperoxides which
are then further reduced by GSH.

PUFA-O-O + Toc-OH PUFA-O-O-H + Toc-O

Toc-O is a stable radical and oxidized form of tocopherol. It is partially reduced again by
ascorbate or GSH.

Cartenoids:

24


Another lipophilic antioxidant are cartenoids that belong to a group called tetraterpenoids,
which means that they are compounds with 40 carbons. Cartenoids can also be further divided
depending on if the molecule contain oxygen or not:
- Xantophylls: Cartenoids containing oxygen, e.g. lutein.

- Carotenes: The unoxygenated form of carotenoids, e.g. lycopene that is especially
rich in tomatoes that have been processed and cooked. Other examples are -carotene.
Cartenoids are especially rich in green leafy vegetables, and yellow, orange, and red
friuts and vegetables. They also, like tocopherols, reduce peroxyl radicals and they can
also quench (=undertrycka) singlet oxygen.

Ubiquinol QH
2
:

Ubiquinol occurs in all cell membranes and regenerates tocopherol in its reduced form QH
2
.

Toc-O + QH
2
Toc-OH + QH

Exogenous sources of ubiquinol are mainly meat and liver. Endogenous production occurs
from the intestinal microflora where the benzoquinone residue is derives from tyrosine and
the isoprenoid chain from farnesyl diphosphate from cholesterol.

18. Naturally-occurring tensides (structural types, micelles, biomembranes,
tensides in lipid digestion):

Naturally occurring tensides:

Normally, lipids contain only non-polar groups that make them insoluble in polar solutes like
water. However, fatty acids, phospholipids, sphingolipids, bile salts, and to a lesser extent,
cholesterol, contain polar groups. This means that these compounds have one polar part and
one non-polar part that make these compounds amphipatic. In water they cluster together to
form micelles that have their non-polar part directed towards the interior part of the
aggregation and the polar soluble part oriented towards the water molecules. A bilayer of such
amphipatic lipids is the basic structure of biological membranes. The formation of micelles
occurs only when there is a certain critical concentration of these amphipatic molecules.
Liposomes are bubbles made up by a lipid bilayer membrane, the same as biological
membranes. They act as carriers of drugs in the circulation that are targeted to specific tissues.
Tensides lower the surface tension by accumulation at the interface of the phases liquid/liquid
and liquid/gas.
Emulsions are much larger particles formed usually by non-polar lipids in aqueous medium
that are stabilized by emulsifying agents that are usually amphipatic lipids, e.g. lecithin.

There are four main types of tensides:

25

- Anionic surfactants: bile salts, soap, fatty acid anions
- Cationic surfactants: quaternary ammonium salts
- Amphoteric surfactants: phospholipids, proteins, betaine
- Non-ionic surfactants: higher alcohols, monoacylglycerols

Bile salts are very important in lipid digestion because they aggregate into micelles and
liposomes and form mixed micelles with the products of fat digestion and are important in
facilitating absorption of lipids from the intestine. Bile salts also neutralize the acidic chyme
that is the bolus coming from the pylorus of the stomach containing hypochloric acid,
digestive enzymes, water and partially digested food.

19. The citric acid cycle - localization, reactions of the cycle; the amphibolic
role of the cycle (the final pathway for the oxidation of nutrients, and the
pathways originating from the cycle):

The citric acid cycle:

The citric acid cycle or tricarboxylic acid cycle is an aerobic process located in the matrix of
mitochondria in eukaryotes. It is a part of the metabolic pathway where lipids, proteins and
carbohydrates are converted into water and CO
2
to generate useful energy. The H atoms are
taken care of by cofactors that transport these to the respiratory chain to contribute to the
oxidative phosphorylation. The main purpose of the citric acid cycle is to reduce as many
cofactors as possible and give rise to NADH and FADH
2
that can drive the respiratory chain
by oxidation of carbon units. Another main purpose is to provide substrates for amino acid
synthesis by transamination, for gluconeogenesis, and for fatty acid synthesis. Because of
these two main purposes, both oxidative and synthesizing processes, the citric acid cycle is
said to be amphibolic. The citric acid cycle is the final common pathway for oxidation of all
major nutrients. Necessary vitamins for the citric acid cycle are: riboflavin, niacin, thiamin,
and pantothenic acid.
The citric acid cycle involved 8 reactions with the initiating reaction of oxaloacetate and
acetyl-CoA. Fatty acids, proteins and carbohydrates have been metabolized to acetyl-CoA.
See separate paper for all reactions of the citric acid cycle. Almost all reactions are reversible,
except 3 reactions.

Sources of acetyl-CoA involve:
- Oxidative decarboxylation of pyruvate from glucose and some AA
- -oxidation of fatty acids
- Catabolism of some amino acids (Threonine, Tryptophan, Lysin, Leucine,
Isoleucine)
- Ketone body utilization in extrahepatic tissues:


26

acetoacetate acetoacetyl-CoA acetyl-CoA

- Catabolism of ethanol acetaldehyde acetate acetyl-CoA

20. The energetic yield and regulation of the citric acid cycle. The
anaplerotic reactions (replenishing the intermediates of the cycle):

The energetic yield:

Acetyl-CoA + 3 NAD
+
+ FAD + 2 H
2
O + H
+
+ HPO
4
2-
+ GDP

2 CO
2
+ CoA-SH + 3 NADH + 3 H
+
+ FADH
2
+ GTP

- 2 C atoms are completely oxidized to 2 CO
2


- 8 H atoms are released in the form of reduced cofactors (3 NADH + H
+
, 1 FADH
2
)

1 GTP = 1 ATP (respiratory chain)

3 NADH + H
+
= 9 ATP (respiratory chain)

1 FADH
2
= 2 ATP (respiratory chain)

Regulation of the citric acid cycle:

The respiratory chain and the oxidative phosphorylation regulates the citric acid cycle. Thus,
the activity is directly dependent on the amount of oxidized NAD
+
that is able to accept H
atoms. The concentration of NAD
+
is in turn dependent on the availability of ADP that is
dependent on the rate of utilization of ATP by chemical and physical work. The citric acid
cycle is largely determined by substrate availability and product inhibition. In addition to this,
the citric acid cycle is also regulated by regulation of the individual enzymes of it. Enzymes
that are regulated are: pyruvate dehydrogenase, citrate synthase, isocitrate
dehydrogenase, and -ketoglutarate dehydrogenase. These enzymes participate in
irreversible reactions.
NADH is a product of all dehydrogenases, except for succinate dehydrogenase and it inhibits
pyruvate dehydrogenase, isocitrate dehydrogenase, -ketoglutarate dehydrogenase, and citrate
synthase.
Acetyl-CoA inhibits pyruvate dehydrogenase, while succinyl-CoA inhibits succinyl-CoA
synthetase and citrate synthase.
ATP inhibits citrate synthase, -ketoglutarate dehydrogenase, and isocitrate dehydrogenase.
ADP is an allosteric activator of these enzymes.
Calcium is another regulator that activates pyruvate dehydrogenase, isocitrate dehydrogenase,
and -ketoglutarate dehydrogenase. Calcium concentrations increase during muscle

27

contraction when the demand for energy increases. Ca
2+
increases many of the steps in the
citric acid cycle and increases the flux throughout the whole pathway.
Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme
involved in glycolysis that catalyzes the formation of fructose-1,6-bisphosphate, a precursor
of pyruvate. This prevents a constant high flux when there is an accumulation of citrate or a
decrease in substrate for the enzyme.
Another vital inhibition of the citric acid cycle is oxygen since the citric acid cycle can only
occur when oxygen is present due to that reduced cofactors must be reoxidized in the
respiratory chain and when this is not possible they inhibit the citric acid cycle.

The anaplerotic reactions:

Anaplerotic reactions of the citric acid cycle are reactions that form intermediates of the cycle.
The intermediates are not only used in the citric acid cycle but also take part in other
pathways and reactions (cataplerotic reactions) and the purpose of the anaplerotic reactions
is to replenish (=terfylla) the intermediates and assure constant concentrations of the
intermediates are available to make the citric acid cycle proceed. It is crucial that the
anaplerotic flux equals the cataplerotic flux.
There are 4 major reactions classed as anaplerotic:
1. Pyruvate Oxaloacetate:

Pyruvate + H
2
O + CO
2
+ ATP Oxaloacetate + ADP + P
i
+ 2 H
+


This reaction is catalyzed by pyruvate carboxylase that is activated by acetyl-CoA,
indicating a lack of oxaloacetate. Pyruvate can also be converted into L-malate in a
similar way by reductive carboxylation. If acetyl-CoA accumulates it acts both as an
allosteric activator of pyruvate carboxylase and inhibitor of pyruvate dehydrogenase.
Lactate, an important substrate for gluconeogenesis, enters the cycle by oxidation to
pyruvate and then carboxylation to oxaloacetate. When there are large amounts of
oxaloacetate it can enter gluconeogenesis by phosphoenolpyruvate carboxykinase that
converts it into phosphoenolpyruvate.

2. Aspartate Oxaloacetate:

This is a reversible reaction forming oxaloacetate from aspartate in a transamination
reaction, via aspartate transaminase.

3. Glutamate -ketoglutarate:

Glutamate + NAD
+
+ H
2
O -ketoglutarate + NH
4
+
+ NADH + H
+


This reaction is catalyzed by glutamate dehydrogenase.

4. Propionyl-CoA Succinyl-CoA: When odd-chain fatty acids are oxidized, one

28

molecule of succinyl-CoA is formed per fatty acid. The final enzyme is
methylmalonyl-CoA mutase.
Other anaplerotic reactions involve catabolism of tyrosine and phenylalanine generates
fumarate. Also, aspartate in the synthesis of urea and purines also generate fumarate.
Besides from the -oxidation, succinyl-CoA is also generated by the catabolism of valine,
methionine, and isoleucine.
Aminotransferases (transaminase) reactions form pyruvate from alanine, oxaloacetate from
aspartate, -ketoglutarate from glutamate. Because all these reactions are reversible, the cycle
also serves as a source of carbon skeletons for synthesis of amino acids. Other amino acids
contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle
intermediates.
- Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield
pyruvate.
- Arginine, histidine, glutamine, and prolin yield -ketoglutarate.
- Isoleucine, methionine, and valine yield succinyl-CoA
- Tyrosine, aspartate and phenylalanine yield fumarate

Acetyl-CoA is the major substrate for fatty acid synthesis and it is formed from pyruvate by
pyruvate dehydrogenase. Pyruvate dehydrogenase is a mitochondrial enzyme and fatty acid
synthesis is a cytosolic pathway and the mitochondrial membrane is impermeable to acetyl-
CoA. Thus, acetyl-CoA is available as citrate is formed, then transported to the cytosol and
cleaved in a reaction catalyzed by ATP-citrate lyase. Citrate is only available for transport
out if the mitochondrion when aconitase is saturated with its substrate and citrate cannot be
channeled directly from citrate synthase to aconitase. This ensures that citrate is used for fatty
acid synthesis only when there is an adequate amount to ensure continued activity of the
cycle.

21. Transport of glucose into cells. Glucose transporters types:

Glucose is an essential substrate for the metabolism of most cells. Because glucose is a polar
molecule, transport through biological membranes requires specific transport proteins. For
this purpose there are glucose transporters that are integral membrane. Each type of glucose
transporter plays a specific role in metabolism. There are 2 main types of glucose transporters;
glucose transporters, and sodium-coupled glucose transporters. GLUT1-14 have 12
helices and work by the mechanism of facilitated diffusion along the concentration gradient
that does not require energy. The different isoforms of glucose transporters have differences
in:
- Affinity to glucose
- Different way of regulation
- Tissue specific occurrence


When the glucose transporter binds to glucose on the outer side of the membrane, this action

29

alters conformational changes so that it cannot bind another glucose molecule. Then glucose
is channeled to the inside of the membrane. In total there ate two conformational changes of
the transporter. This action can be dependent on insulin and have different affinities for
glucose to occur.

GLUT1: It is distributed in fetal tissue, in adults; it is present in erythrocytes and in epithelial
cells of blood-brain barrier. It is responsible for the low-level basal uptake of glucose in cells
to sustain respiration in all cells. The expression of GLUT1 is increased by reduced glucose
levels and decreased by increased glucose levels.
GLUT2: It occurs e.g. in the liver, small intestine, kidney and -pancreatic cells. All
monosaccharides are transported from the intestinal cells into the portal circulation by
GLUT2 and renal glucose reabsorption. It has a high capacity but low affinity for glucose in
pancreatic cells. It transports glucose by passive diffusion and ensures a rapid uptake or
release of glucose.
GLUT3: It is present in the brain, kidney and placenta. It is a high-affinity form of glucose
transporters type I and is believed to be the main glucose transporter for neurons.
GLUT4: It is found in adipose tissue and striated muscle (cardiac, skeletal muscle). The
glucose-uptake is insulin-stimulated. In the absence of insulin, GLUT4 is located in the
interior of adipocytes and striated muscle cells and when activated by insulin it is translocated
to the plasma membrane. At the plasma membrane is allows glucose to be transported by
facilitated diffusion down the concentration gradient. Contraction stimulates the translocation
of the GLUT4 to the cell surface, which explains why it occurs in striated muscle.

Sodium-glucose transport proteins: This type of glucose transporters can be found in the
intestinal mucosa of the small intestine and renal tubules of nephrons. They contribute to renal
glucose reabsorption. These proteins use energy from a downhill sodium gradient to transport
glucose across an uphill concentration gradient. This is an example of secondary active
transport and the sodium-glucose transporters are symporters since both sodium and
glucose are transported together in the same direction across the membrane.

22. The glycolytic pathway - localization, the reactions of glycolysis,
regulation of glycolysis:

The glycolytic pathway:

The glycolysis is located in the cytosol of all eukaryotic cells and is the major pathway for
glucose metabolism. It is unique in the sense that it can both work aerobically and
anaerobically depending on the presence of oxygen and a respiratory chain. Erythrocytes are
totally dependent on glucose metabolism as a source of energy since they lack mitochondria
that perform oxidative phosphorylation. Glycolysis in erythrocytes is anaerobic due to this.
Glycolysis is the main pathway for glucose, but also for fructose, galactose and other
carbohydrates obtained from the diet. It is very important that the glycolysis can be run
anaerobically since this makes tissues less susceptible to anoxic episodes and it provides a fast
extra energy supply for skeletal muscles when the supply of oxygen is not sufficient for the
high performance required. The overall reaction formula of glycolysis is as follows:

30


Glucose + 2 ADP + 2 P
i
+ 2 NAD
+
2 Pyruvate + 2 ATP + NADH
1. Glucose + ATP Glucose-6-Phosphate + ADP + P
i


The phosphorylation of glucose traps the glucose inside the cell because glucose-6-
phosphate is negatively charged and thus cannot diffuse through the membrane. The
addition of phosphoryl to glucose makes it more instable which facilitates further
metabolism. This reaction is irreversible, but in the kidney and liver the reverse
reaction, catalyzed by glucose-6-phosphatase, is also able where glucose is released
into the bloodstream.
The formation of glucose-6-phosphate is catalyzed both by hexokinase and
glucokinase. Hexokinase occurs in all tissues and has higher affinity for glucose than
glucokinase that only occurs in the kidney and liver and that has lower affinity for
glucose. Glucokinase is also specified to glucose, while hexokinase catalyzes the
conversion of all hexoses. In addition, glucokinase is not inhibited by its products,
glucose-6-phosphate, hexokinase though is inhibited by it in tissues where glucokinase
is present. High concentrations of hexokinase signalizes that the cell no longer needs
glucose for energy, and instead it is stored as glycogen.

2. Glucose-6-Phosphate Fructose-6-Phosphate

An isomerisation of glucose-6-phosphate to form fructose-6-phosphate. This reaction
is catalyzed by phosphohexose isomerase.

3. Fructose-6-Phosphate + ATP Fructose-1,6-Bisphosphate + ADP

This phosphorylation reaction is catalyzed by phosphofructokinase and the rate of
this reaction determines the rate of the whole glycolysis. Phosphofructokinase is
allosterically inhibited by ATP and citrate and allosterically activated by ADP, AMP
and fructose-2,6-bisphosphate in the liver.

4. Fructose-1,6-bisphosphate Dihydroxyacetonephosphate
Glyceraldehyde-3-phosphate

Fructose-1,6,-bisphosphate is cleaved by aldolase into two triose phosphates.
Glyceraldehyde-3-phosphate and dihydroxyacetonephosphate are interconvertable by
phosphotriose isomerase.

5. Glyceraldehyde-3-Phosphate +P
i
+ NAD
+
1,3-Bisphosphoglycerate + NADH
+ H
+

This reaction is catalyzed by glyceraldehydes-3-phosphate dehydrogenase and is
inhibited by iodoacetate. Important to note is that the incoming phosphate does not
originate from ATP, but from inorganic phosphate from the surrounding environment.
The phosphoryl group attached to glyceraldehydes-3-phosphate has an ester bond,
while the incoming phosphoryl group that attached to the anhydride forms an
anhydride bond.

31



6. 1,3-Bisphosphoglycerate + ADP + P
i
3-Phosphoglycerate + ATP

This reaction generates ATP by substrate-level phosphorylation and is catalyzed by
phosphoglycerate kinase. In erythrocytes 1,3-Bisphosphoglycerate has a side
reaction where it is converter to 2,3-Bisphosphoglycerate by mutase that is important
in the release of oxygen in oxygenated hemoglobin. 2,3-Bisphosphoglycerate is then
converted to 3-Phosphoglycerate + P
i
by 2,3-bisphosphoglycerate phosphatase and
can join the glycolysis again.

7. 3-Phosphoglycerate 2-Phosphoglycerate

This isomerization reaction is catalyzed by phosphoglycerate mutase.

8. 2-Phosphoglycerate Phosphoenolpyruvate + H
2
O

This dehydration reaction is catalyzed by enolase and is inhibited by F
-
. This is taken
advantage of in blood sampling where the blood contained in tubes containing F
-
that
will inhibit glycolysis so that the actual concentration of glucose in the blood can be
measured.

9. Phosphoenolpyruvate + ADP + P
i
Pyruvate + ATP

ATP is formed from substrate-level phosphorylation catalyzed by pyruvate kinase in
an irreversible reaction. Pyruvate kinase is activated by fructose-1,6-bisphosphate
and inhibited by glucagon.

Regulation of glycolysis:

The glycolysis is regulated at 3 steps involving non-equilibrium reactions. These three
reactions are catalyzed by hexokinase (and glucokinase), phosphofructokinase, and
pyruvate kinase. In gluconeogenesis, different enzymes are used at these three sites.
Phosphofructokinase is inhibited by ATP. Fructose enters the glycolysis by phosphorylation
to fructose-6-bisphosphate and thus bypasses main regulatory steps so that it can form more
pyruvate (and acetyl-CoA) than required for ATP formation. In adipose tissue and liver this
leads to increased lipogenesis and therefore fructose can contribute to obesity.
In erythrocytes 1,3-bisphosphoglycerate is converted to 2,3-bisphosphoglycerate that is
important in the release of oxygen from hemoglobin. This pathway generates no net yield of
ATP.
Hexokinase is inhibited by citrate, acetyl-CoA and glucose-6-phosphate, except in the liver.
Phosphofructokinase is allosterically activated by fructose-2,6-bisphosphate and inhibited
by glucagon and epinephrine.
Pyruvate kinase is allosterically activated by phosphoenolpyruvate and fructose-1,6-

32

bisphosphate. It is allosterically inhibited by ATP and also by alanine.

23. The glycolysis under anaerobic conditions - the role of lactate
dehydrogenase reaction, the Cori cycle; the LD Isoenzymes:

Anaerobic glycolysis is run when oxygen is deficient or limited. It works as an efficient way
of fast generation of ATP but only for a short period of time. 2 ATP per glucose molecule can
be achieved which is formed much faster than that of oxidative phosphorylation which is very
valuable in threatening situations when skeletal muscles need fast extra energy supply.
In aerobic glycolysis glucose from glycogen stores is routed to produce lactate instead of
pyruvate. The lack of oxygen results in a paucity (=brist) of oxidized NAD
+
since it needs a
H
+
acceptor which is the role of oxygen (mitochondrial respiratory chain). This is solved by
pyruvate being oxidized to lactate by lactate dehydrogenase that also oxidized NADH and
thus generates NAD
+
that can again accept H
+
. Concentration of lactate in blood is about 1,0
mmol/l.

Pyruvate + NADH + H
+
Lactate + NAD
+


Glucose + 2 ADP + P
i
2 Lactate + 2ATP + 2 H
2
O

In anaerobic glycolysis the amount of ATP formed per mole of glucose oxidized is limited, so
that much more glucose must be metabolized during anaerobic conditions than aerobic
conditions. In yeast and some microorganisms, pyruvate is not reduced to lactate but reduced
and decarboxylated to ethanol.

Lactate dehydrogenase Isoenzymes:

Lactate dehydrogenase catalyzes the reduction of pyruvate to lactate and the reverse reaction
where lactate is oxidized to pyruvate. The lactate dehydrogenase has 5 isoforms (LD1-5) each
with 4 subunits; either M or H subunits. H subunits are typical for cardiac muscle and M
subunits for skeletal muscle.

The Cori cycle:

The cori cycle is the metabolic pathway where lactate produced in anaerobic glycolysis in
muscles is transported to the liver where it is converted into glucose and returned to the
muscles again where it can enter glycolysis again either anaerobically or aerobically to
become lactate or pyruvate. The uptake of lactate of the liver is crucial since lactate is acidic
and can potentially lead to lactic acidosis if accumulated. Lactate that enters the liver is first
converted into pyruvate by the same enzyme as in muscle, lactate dehydrogenase, and then
into glucose by the gluconeogenesis. Then the glucose can enter the muscles again via the
bloodstream.

24. The energetic yield of glycolysis under anaerobic and anaerobic
conditions:

The yield in ATP by substrate-level phosphorylation for the anaerobic -and aerobic glycolysis

33

is consumption of 2 ATP and production of 4 ATP yielding 2 ATP/glucose in profit. This is
the only yield in anaerobic glycolysis.

Further yield of aerobic glycolysis is;
- 2 NADH in the oxidative phosphorylation of glyceraldehydes-3-phoshate to 1,3-
bisphosphoglycerate. 2 x 2 ATP
- Conversion of pyruvate to acetyl-CoA (2NADH) 2 x 3 ATP
- Conversion of acetyl-CoA in the citric acid cycle (2 NADH) 2 x 12 ATP

Total energy yield of aerobic glycolysis: Aerobic glycolysis till pyruvate:
Glucose 2 Pyruvate : 2 ATP (substrate phosphorylation), 2 NADH 4-6 ATP in RC

Further conversions of pyruvate:

2 Pyruvate 2 Acetyl-CoA + 2 NADH: 6 ATP (2x NADH to the respiratory chain)

2 Acetyl-CoA 2 CO
2
+ 6 NADH + 2 FADH
2
: 2 x 12 ATP = 24 ATP

TOTAL YIELD OF AEROBIC GLYCOLYSIS: 36-38 ATP

25. The oxidative decarboxylation of pyruvate and other 2-oxoacids
(localization, the roles of particular coenzymes in the pyruvate and 2-
oxoglutarate dehydrogenase complexes, energetics, significance):

The oxidative decarboxylation of pyruvate:

Pyruvate formed in the glycolysis in the cytosol under aerobic conditions is transported into
the matrix of the mitochondria where enzymes of the citric acid cycle are located. Pyruvate is
transported by a proton symporters into the mitochondria. Inside the mitochondrion it is
oxidatively decarboxylated by a pyruvate dehydrogenase complex consisting of 3
enzymes that is analogous to the -ketoglutarate dehydrogenase complex in the citric acid
cycle. Cofactors necessary for pyruvate dehydrogenase activity are thiamine diphosphate
(TDP), lipoate, coenzyme A, FAD, and NAD
+
.
1. Decarboxylation of pyruvate: In the first reaction pyruvate is decarboxylated by the
thiazolium ring of thiamine diphosphate which forms hydroxyethyl-TDP.

2. Transfer of acetyl to lipoate: Hydroxyethyl-TDP in turn reacts with oxidized lipoamide
(prosthetic group of dihydrolipoyl transacetylase), to form acetyl lipoamide. The
hydroxyethyl group is dehydrogenated to thioester during transfer. One of the H atoms
reduced sulphur atom of lipoate to SH. The second H atom goes back to thiamine
diphosphate. Thiamine is a vitamin B
1
and in deficiency this pathway is impaired which leads
to accumulation of pyruvate or lactate that can lead to lactic or pyruvic acidosis.

3. Transfer of acetyl to coenzyme A: Acetyl lipoamide reacts with coenzyme A to form

34

acetyl-CoA and reduced lipoamide.

4. Transfer of 2 H
+
to NAD
+
via FAD: The reaction is completed when the reduced
lipoamide is reoxidized by a flavoprotein, dihydrolipoyl dehydrogenase, containing FAD.
Finally, the reduced flavoprotein is oxidized by NAD
+
which is transferred to the respiratory
chain in its reduced form.
Pyruvate + NAD
+
+ CoA Acetyl-CoA + NADH + H
+
+ CO
2


Oxidative decarboxylation of 2-oxo-acids:

Another name for oxo acids is keto acids which are inorganic acids containing a ketone
functional group and a carboxylic group. 2-oxo acids or -keto acids have a keto group
adjacent to the carboxylic group. In this way, according to the distance from the keto group
and the carboxylic group we can divide keto acids into , , etc.
Important 2-oxo acids are e.g.; -ketoglutarate (2-oxoglutarate), and oxaloacetate

-Ketoglutarate:

Oxaloacetate:

26. Glycogen synthesis - localization, reactions, control mechanisms:

Glycogen synthesis:

Glycogen is the major storage carbohydrate in animals, corresponding to starch in animals.
Glycogen is a branched polymer of -D-glucose that occurs mainly in the liver and muscles.
The branched structure of glycogen increases its solubility in water and allows enzymes to
work on several branched simultaneously. The main chain are connected via -1,4-glycosidic
bonds, while the branches are attached to the main chain via -1,6-glycosidic bonds.
Muscle glycogen provides a readily available source of glucose for glycolysis within the
muscle itself. Liver glycogen serves to store and export glucose to maintain blood glucose
between meals. Glycogen is stored in the cytoplasm of cells in the form of glycogen particles
and enzymes involved in its synthesis and degradation are located on the surface of these
granules. It is important to note that glycogenolysis is not a reverse process of glycogen
synthesis. Liver concentrations of glycogen is about:

After meal: 450 mM
Overnight fast: 200 mM
After 12-18 h of fasting: Glycogen stores almost depleted

Muscle tissue lacks the enzyme glucose -6-phosphatase that transforms glycogen into
glucose. Therefore, pyruvate formed from glycolysis is transaminated to alanine which is
exported from the muscle to the liver where it is used in gluconeogenesis.

Synthesis of glycogen involves 4 main steps:

35


1. Activation of glucose into UDP-glucose:
First of all glucose of phosphorylated to glucose-6-phosphate by hexokinase or
glucokinase. Then glucose-6-phosphate is isomerised to glucose-1-phosphate by
phosphoglucomutase. Next step involves activation of glucose-1-phosphate into
UDP-glucose by UDP-Glc-pyrophosphatase. This activation takes place by glucose-
1-phosphate reacting with UTP. This reaction proceeds in the direction of UDP-Glc
formation because it catalyzes the hydrolysis of pyrophosphate (PP
i
) into 2 P
i
and thus
it removes one of the reaction products.

2. Primer is necessary for synthesis of glycogen:
A pre-existing glycogen molecule, or a primer, must be present to initiate the
synthesis of glycogen. The glycogen primer is formed on a protein primer known as
glycogenin that is glycosylated on a tyrosine residue by UDP-Glc. The use of a
glycogen primer is only used when all glycogen stores are depleted and no glycogen
molecule is present to act as the start point of glycogen synthesis.

3. Formation of -1,4-glycosidic bonds:
This process can be further divided:
- Initiation: Glycosyl residue is added from UDP-Glc (C1) to the non-reducing end of
the primer (C4) by glycogen synthase. UDP is released by this action and it serves as
a carrier of glucose molecules.
- Elongation: Elongation is performed by glycogen synthase and glucose residues are
attached in the 1 4 direction forming -1,4-glycosidic bonds.

4. Branching:
When the chain is at least 11 glucose residues long, branching enzyme transfers a
part of the 1 4 chain (5-8 glycosyl residues long) to neighbouring chain to form -
1,6-glycosidic bonds, establishing a branch point. The branches grow by further
additions of 1 4 glycosyl units to the branch and further branching.

Control mechanisms:

Glycogen synthase is regulated by allosteric mechanisms and covalent modifications by
reversible phosphorylation and dephosphorylation of the enzyme protein in response to
hormone action. Phosphorylation of glycogen synthase is increased in response to cyclic
AMP (cAMP) formed from ATP by adenylyl cyclase at the inner surface of the cell
membrane in response to hormones such as epinephrine, norepinephrine, and glucagon.
cAMP is hydrolyzed by phosphodiesterase and by that terminating hormone action; in the
liver insulin increases the activity of phosphodiesterase.
From this, glycogen synthase exists in both phosphorylated and non-phosphorylated states.
Active glycogen synthase a is dephosphorylated and inactive glycogen synthase b is
phosphorylated. The opposite relations are valid for phosphorylase. Dephosphorylation is

36

performed by phosphatases, phosphorylation by kinases and ATP.
6 different protein kinases act on glycogen synthase, 2 of them are Ca
2+
/calmodulin
dependent. Another kinase is cAMP-dependent protein kinase, which allows cAMP-
mediated hormonal action to inhibit glycogen synthesis synchronously with the activation of
glycogenolysis. Insulin promotes glycogenesis by raising glucose-6-phosphate levels, which
stimulates the dephosphorylation and activation of glycogen synthase. Dephosphorylation of
glycogen synthase b is carried out by protein phosphatase-1 which is under control of
cAMP-dependent protein kinase.

27. Glycogenolysis in the liver and skeletal muscle - the steps and control of
glycogen degradation, inherited disorders:

Glycogenolysis:

Glycogenolysis is the degradation of glycogen that proceeds during fasting (liver), muscle
work (muscle) or stress (liver + muscle). Glycogenolysis is not the reverse process of
glycogenesis, but a separate pathway. Glycogen phosphorylase catalyzes the rate-limiting
step in by catalyzing the phosphorolytic cleavage (cleavage of a compound in which
inorganic phosphate is the attacking group) of the 1 4 linkages of glycogen to yield
glucose-1-phosphate. Glycogen phosphorylase requires pyridoxal phosphate as its
coenzyme. The phosphate group of phosphorylase is the active group of the enzyme and this
explains why there is a phosphorolytic cleavage. Finally, the glycosyl residues are removed
from the main chain until about 4 residues are left around each branch. Hydrolysis of the
branches is finally completed by debranching enzyme. Here is a more detailed description:


1. Phosphorolytic cleavage of -1,4-glycosidic bonds by phosphorylase:

A phosphorolytic cleavage by phosphorylase is performed at the non-reducing ends until
about 4 glycosyl residues remain before a branching point on the 1,6 branch, by this limit
dextrins are formed.

2. Removal of -1,6 branching by debranching enzyme:

Debranching enzyme consists of two enzymes involved in the degradation of the 1,6
branches; transferase and glucosidase.
Transferase transfers 3 out of the 4 glycosyl residues of the limit dexrin and adds these to the
main chain by -1,4-glycosidic bonds.
Glucosidase hydrolyses the remaining glycosyl residue at the 1,6 branch by 1,6-glucosidase
activity of the debranching enzyme. By this free glucose is released, not glucose-1-phsophate.
By making the branched chain linear phosphorylase can cleave the whole chain.

The glucose-1-phosphate formed from the phosphorolytic cleavage of glycogen can be
converted to glucose-6-phosphate by the enzyme phosphoglucomutase. Glucose-6-
phosphate can then enter the glycolysis and serve as fuel for further ATP synthesis. In the

37

liver and kidney glucose-6-phosphate can be converted into free glucose by glucose-6-
phosphatase by hydrolysis. Glycogen-6-phosphatase is located in the ER of liver and kidney
cells. Glucose-6-phosphate is impermeable and only glucose can diffuse through the cell
membrane, thus, cleavage of muscle glycogen cannot serve as regulation of blood glucose.





Regulation of glycogenolysis:

Glycogen phosphorylase is also regulated by allosteric mechanisms and covalent
modifications by phosphorylation and dephosphorylation in response to hormone action.
In the liver the role of glycogen is to maintain the blood concentrations of glucose; in muscle
it serves to provide glucose-6-phosphate for glycolysis in response to the need for ATP during
muscle contraction. In both tissues, phosphorylase is activated by phosphorylation
catalyzed by phosphorylase kinase (to yield phosphorylase a), and inactivated by
dephosphorylation by protein phosphatase (to yield phosphorylase b) in response to
hormones and other signals.
Active liver phosphorylase a is allosterically inhibited by ATP, glucose-6-phosphate, and
free glucose.
Muscle phosphorylase differs from the liver isoenzyme in having a binding site for 5AMP
that acts as an allosteric activator of the inactive dephosphorylated form of phosphorylase b.
5AMP acts as a potent signal of the energy state of the muscle; it is formed as the
concentration of ADP begins to increase which signals that the substrate metabolism must be
increased to permit ATP formation.
Phosphorylase kinase is activated in response to cAMP. Phosphorylase kinase is dependent
on cAMP and it catalyzes the phosphorylation by ATP of inactive phosphorylase kinase b to
active phosphorylase kinase b. In the liver, cAMP is formed in response to glucagon, which
is secreted in response to falling blood glucose. Muscle is insensitive to glucagon; in muscle,
the signal for increased cAMP is the action of norepinephrine which is secreted in response
to fear of fright; when there is a need for increased glycogenolysis to permit rapid muscle
activity.
Increased concentrations of cytosolic Ca
2+
is responsible for both initiation of both
contraction and glycogenolysis in muscle.
In the liver, glycogenolysis is activated independently by cAMP, but in response to
epinephrine and norepinephrine. This involves mobilization of Ca
2+
into the cytosol,
followed by stimulation of a Ca
2+
/calmodulin-sensitive phosphorylase kinase. cAMP-
independent glycogenolysis is also activated by vasopressin, oxytocin and angiotensin II
acting either through calcium or the phosphatidylinositol bisphosphate pathway.

Both phosphorylase a and phosphorylase kinase a are dephosphorylated by protein
phosphatase-1. Protein phosphatase-1 is inhibited by inhibitor-1, which is active only after it
has been phosphorylated by cAMP-dependent protein kinase. Thus, cAMP controls both the

38

activation and inactivation of phosphorylase. Insulin reinforces this effect by inhibiting the
activation of phosphorylase b. It does this indirectly by increasing uptake of glucose, leading
to increased formation of glucose-6-phosphate, which in turn is an inhibitor of phosphorylase
kinase.
At the same time as phosphorylase is activated by a rise in concentration of cAMP (via
phosphorylase kinase), glycogen synthase is converted into the inactive form. Both effects are
mediated by cAMP-dependent protein kinase. Thus, inhibition of glycogenolysis enhances
net glycogenesis. The dephosphorylation of phosphorylase a, phosphorylase kinase, and
glycogen synthase b is catalyzed by protein phosphatase-1. In turn, protein phosphatase is
inhibited by cAMP-dependent protein kinase via inhibitor-1.
Inherited disorders:

Glycogen storage diseases is a generic term referring to a group of inherited disorders
characterized by deposition of abnormal type or quantity of glycogen in tissues or failure to
mobilize glycogen.

Von Gierke disease (type I):
It is the most common glycogen storage disease where the liver is deficient in glucose-6-
phosphatase or transporters for this enzyme. The reaction where glucose-6-phosphate is
converted into glucose is impaired. Consequences of this disorder includes hypoglycaemia at
fasting, lactacidemia, and might also cause hyperlipidemia and hyperurikemia.

Pompe disease (type II):

In the lysosomes, the enzyme -1,4-glucosidase is absent that is involved in the debranching
in glycogenolysis. This leads to an accumulation of glycogen in lysosomes and the loss of
their function. Muscles are damages by this and they become weak. Pompe disease occurs in
an adult, juvenile and infant form. Infants die before the age of 2, in juvenile form it causes
myopathy and in adults it gives rise to muscular dystrophy.

McArdle disease (type V):

There is an absence of muscular phosphorylase which impairs the breakdown of the
glycogen stores that cannot be utilized because of this. This leads to that the muscles are not
able to perform exercise or work.

28. Gluconeogenesis (localization, substrates and the course of
gluconeogenesis, regulation), relationship between glycolysis and
gluconeogenesis:

Gluconeogenesis:

Concentration of glucose in blood: 3,1-5,0 mmol/l

Gluconeogenesis is the process of synthesizing glucose or glycogen from non-carbohydrate
precursors. Major substrates are glucogenic amino acids, lactate, glycerol and propionate.
Liver and kidney are major gluconeogenic tissues (cytosol of cells), but the small intestine

39

may also be a source of glucose in the fasting state.
Gluconeogenesis meets the need for glucose in the fasting state when carbohydrates are
insufficient. A supply of glucose is especially necessary for erythrocytes and the nervous
system. Hypoglycemia causes brain dysfunction that in extreme cases can lead to coma or
even death.
Glucose is also important in maintaining the intermediates of the citric acid cycle even when
fatty acids are the main source of acetyl-CoA in the tissues. In addition, gluconeogenesis
clears lactate produced by muscle and erythrocytes and glycerol produced by adipose tissue.
Enzymes of the glycolysis are also used in gluconeogenesis, but at three irreversible reactions
other enzymes are used. Lactate can be used as substrate for gluconeogenesis by the Cori
cycle where it is transported to the liver where it is converted back into pyruvate by lactate
dehydrogenase. Pyruvate, which is the first substrate in gluconeogenesis, can then be used to
generate glucose.
All citric acid cycle intermediates, through conversion into oxaloacetate, amino acids (not
Leucine and lysine), and glycerol can serve as fuels for gluconeogenesis. Transamination
and deamination of amino acids facilitates the entering of their carbon skeletons into the
cycle (as pyruvate or oxaloacetate) or indirectly via the citric acid cycle.
Fatty acids cannot be converted into glucose in animals, with an exception of odd-chain fatty
acids, which yield propionyl-CoA, a precursor of succinyl-CoA. Glycerol, which is a part of
triacylglycerols, can also be used in the gluconeogenesis.
Glycerol + ATP Glycerol-3-phosphate
Glycerol-3-phosphate + NAD
+
Dihydroxyacetone + NADH + H
+


It is important to note that acetyl-CoA is not a substrate for gluconeogenesis, it is metabolized
to CO
2
in the citric acid cycle. Fatty acids cannot be converted into glucose in animals.
Gluconeogenesis starts in the mitochondria, or cytoplasm, depending on which substrate is
being used. In total there are 11 steps, where 3 steps are unique to the gluconeogenesis:

1. Synthesis of phosphoenolpyruvate:

The production of phosphoenolpyruvate is an irreversible reaction of the reaction in
glycolysis catalyzed by pyruvate kinase. It involves 2 partial reactions:

- Pyruvate Oxaloacetate: This carboxylation reaction is located in the mitochondria
and catalyzed by pyruvate carboxylase and is an ATP-requiring reaction with biotin
as cofactor. Biotin binds CO
2
from bicarbonate as carboxybiotin prior to the addition
of the CO
2
to pyruvate. This reaction is also an anaplerotic reaction of the citric acid
cycle.

- Oxaloacetate Phosphoenolpyruvate: Phosphoenolpyruvate carboxykinase
catalyzes the decarboxylation and phosphorylation of oxaloacetate to
phosphoenolpyruvate requiring 1 GTP as the phosphate donor. In liver and kidney,
the reaction of succinate thiokinase in the citric acid cycle provides this GTP. This
provides a link between the citric acid cycle and the gluconeogenesis. This prevents

40

excessive removal of oxaloacetate from the citric acid cycle which would prevent it.
The decarboxylation of oxaloacetate occurs partially in the cytoplasm, but since
oxaloacetate is impermeable to the inner mitochondrial membrane it joins the
aspartate/malate shuttle by which it is transported. The reason for that carboxylation
occurs before decarboxylation is that when metabolism is affected by glucagon in
hypoglycemia, fatty acids are released from adipose tissue and oxidized in the -
oxidation into acetyl-CoA. Acetyl-CoA in turn inhibits pyruvate dehydrogenase and
activates pyruvate carboxylase.

2. Fructose-1,6-Bisphosphate Fructose-6-Phosphate:

This dephosphorylation is catalyzed by fructose-1,6-bisphosphatase. It is present in
skeletal muscle, liver and kidney, but absent in heart-and smooth muscle. Its presence
determines whether a tissue is capable of synthesizing glucose (or glycogen) not only
from pyruvate, but also from triose phosphates.
3. Glucose-6-phosphate Glucose:

The conversion of glucose-6-phosphate into glucose is only possible in the liver and
kidney since the enzyme catalyzing this dephosphorylation, glucose-6-phosphatase,
is deficient in muscle and adipose tissue. Water makes a hydrolytic cleavage of the P
i
.
The glucose-6-phosphatase is present in the ER of the liver and kidney.

The total energetic requirement of the gluconeogenesis is 6 ATP, the main source of energy is
from the -oxidation:

2 pyruvate 2 oxaloacetate : 2 ATP
2 oxaloacetate 2 phosphoenolpyruvate : 2 ATP (GTP)
2 3-phosphoglycerate 2 1,3-bisphosphoglycerate: 2 ATP

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H
+
Glucose + 4 ADP + 2 GDP + 6 P
i
+ 2
NAD
+


Regulation of gluconeogenesis:

Gluconeogenesis from lactate and glycerol requires NAD
+
and when the ratio NADH/NAD
+

is high gluconeogenesis from these substrates cannot occur. This occurs e.g. during ethanol
metabolism (alcohol dehydrogenase) and therefore alcoholics are at risk for hypoglycemia
since gluconeogenesis is inhibited.
Since glycolysis and gluconeogenesis share the same pathway but in opposite directions they
have to be reciprocally regulated. One main thing regulating the rate of gluconeogenesis is the
availability of substrates. The availability of substrates is in turn regulated by enzymes that
are regulated by changes in rate of enzyme synthesis, covalent modifications by
phosphorylation/dephosphorylation and allosteric effects.
Insulin enhances the synthesis of key enzymes of glycolysis and simultaneously suppresses

41

gluconeogenesis. It also antagonizes the effect of glucagon-stimulated cAMP, which induces
synthesis of key enzymes of gluconeogenesis.
Glucagon and epinephrine that are responsive to a decrease in blood glucose inhibit
glycolysis and stimulate gluconeogenesis in the liver and kidney by increasing the levels of
cAMP. The increased levels activate cAMP-dependent protein kinase, leading to
phosphorylation and inactivation of pyruvate kinase.

In gluconeogenesis, pyruvate carboxylase that catalyzes the synthesis of oxaloacetate from
pyruvate requires acetyl-CoA as an allosteric activator. The addition of acetyl-CoA results in
a change of the tertiary structure. The activation of pyruvate carboxylase and the reciprocal
inhibition of pyruvate dehydrogenase by acetyl-CoA derived from oxidation of fatty acids
explain the action of fatty acid oxidation in sparing the oxidation of pyruvate and in
stimulating gluconeogenesis.
Phosphofructokinase (phosphofructokinase-1) occupies a key position in regulating
glycolysis and is also subject to feedback control. It is inhibited by citrate and by normal
intracellular concentrations of ATP and is activated by 5AMP. 5AMP acts as an indicator of
the energy status of the cell. In the liver, adenyl kinase allows equilibrium of this reaction:

2 ADP ATP + 5AMP

Thus, when ATP is used in energy-requiring processes resulting in formation of ADP, the
concentration of AMP decreases.
Phosphofructokinase-1 activity is regulated in response to the energy status of the cell to
control the quantity of carbohydrate undergoing glycolysis prior to its entry into the citric acid
cycle. Simultaneously, AMP activates phosphorylase, increasing glycogenolysis. A
consequence of the inhibition of phosphofructokinase-1 is an accumulation of glucose-6-
phosphate, which in turn inhibits further uptake of glucose in extrahepatic tissue by inhibition
of hexokinase.
The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of
fructose-1,6-bisphosphate in liver is fructose-2,6-bisphosphate. It relieves inhibition of
phosphofructokinase-1 by ATP and increases the affinity for fructose-1,6-phosphate.
Fructose-2,6-bisphosphate is formed by phosphorylation of fructose-6-phosphate by
phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown,
since it has fructose-2,6-bisphosphatase activity. This bifunctional enzyme is under
allosteric control of fructose-6-phosphate, which stimulates the kinase and inhibits the
phosphatase. Hence, when there is an abundant supply of glucose, the concentration of
fructose-2,6-bisphosphate increases, stimulating glycolysis by activating
phosphofructokinase-1 and inhibiting fructose-1,6-phosphatase. In the fasting state, glucagon
stimulates the production of cAMP, activating cAMP-dependent protein kinase, which in turn
inactivates phosphofructokinase-2 and activates fructose-2,6-bisphosphatase by
phosphorylation. Hence, gluconeogenesis is stimulated by a decrease in the concentration of
fructose-2,6-bisphosphate, which inactivates phosphofructokinase-1 and relieves the
inhibition of fructose-1,6-bisphosphatase.

The control points in glycolysis and glycogen metabolism involve a cycle of phosphorylation
catalyzed by glucokinase and glucose-6-phosphatase; phosphofructokinase-1 and

42

fructose-1,6-bisphosphate; pyruvate kinase, pyruvate carboxylase, and
phosphoenolpyruvate carboxykinase; and glycogen synthase and phosphorylase. When
glycolysis is enhanced gluconeogenesis is suppressed, however, in muscle there are always
some activity of phosphofructokinase-1 and fructose-1,6-phosphatase at all times to allow
rapid glycolysis at muscle contraction.
When galactose and fructose are ingested they are converted into glucose in the liver via the
hepatic portal vein.
29. The pentose phosphate pathway (localization, the sequence of reactions,
physiological importance):

The Pentose phosphate pathway:

The pentose phosphate pathway is a process that generates NADPH and pentoses (5-carbon
sugars). There are two distinct phases; the first oxidative phase (irreversible reactions)
where NADPH is generated, and the second non-oxidative phase (reversible reactions)
where synthesis of pentoses takes place. The NADPH formed is used for synthesis of fatty
acids and steroids, and ribose is used for synthesis of nucleotides and nucleic acids. The
pentose phosphate pathway serves as an alternative pathway for glucose that does not lead to
the formation of ATP. Fructose and galactose derived from the diet are converted into glucose
in the liver to serve as substrate for the pathway.
The pentose phosphate pathway takes place in liver, adipose tissue, erythrocytes, adrenal
gland, mammary gland, testes, ovaries etc. In all of these tissues the pathway itself is located
in the cytoplasm.

Oxidative part:

See paper for reactions. The total yield of the oxidative phase is 2 NADPH + H
+
and 1
pentose phosphate (ribose).

Non-oxidative part:

See paper for reactions. There are two important enzymes in these reactions; transaldolase
that catalyzes the transfer of 3-carbon units, and transketolase that catalyzes the transfer of
2-carbon units. The reactions of the non-oxidative phase are reversible which enables that
ribose-5-phosphate can be regenerated by intermediates of the glycolytic pathway in case
when the need for nucleotides and nucleic acids is higher than the need for NADPH.

Regulation of the Pentose phosphate pathway:

The pentose phosphate pathway is directed by the cellular need:

NADPH: Oxidative reactions produce NADPH, non-oxidative reactions convert ribulose-5-
phosphate to glucose-6-phosohate to produce more NADPH.

NADPH + Ribose-5-phosphate: Oxidative reactions produce NADPH and ribulose-5-
phosphate, the isomerase converts ribulose-5-phosphate to ribose-5-phosphate.


43

Ribose-5-phosphate: Only the non-oxidative reactions. High level of NADPH inhibits
glucose-6-phosphate dehydrogenase, so transaldolase and transketolase are used to
convert fructose-6-phosphate and glyceraldehydes-3-phosphate into ribose-6-phosphate.

NADPH + Pyruvate: Both the oxidative and non-oxidative reactions are used. The oxidative
reactions generate NADPH and ribulose-5-phosphate, the non-oxidative reactions convert
ribulose-5-phosphate to fructose-6-phosphate and glyceraldehydes-3-phosphate that are
transferred to the glycolysis where they are converted into pyruvate.
The pentose phosphate pathway is important in synthesis of nucleic acids and nucleotides for
which it provides the ribose skeleton. NADPH is important in e.g. reduction of glutathione,
monooxygenase reactions with P450, and reductive syntheses (fatty acid synthesis,
cholesterol synthesis, nucleotide synthesis etc.). It is important to note that the pentose
phosphate pathway is the only source of NADPH for erythrocytes where it is very important
in maintaining the reduced glutathione pool.

30. Synthesis of amino sugars and sialic acids, significance for the synthesis
of glycoproteins and proteoglycans. Synthesis and metabolism of glucuronic
acid (the uronic acid pathway):

Synthesis of amino sugars and sialic acids:

Amino sugars (hexosamines) are sugars that contain an amino group instead of a hydroxyl
group. Several antibiotics contain amino sugars that are important for their antibiotic activity.
The amino sugars include:

- D-Glucosamine (constituent of hyaluronic acid)

- D-Galactosamine (also chrondrosamine)

- D-Mannoseamine

Derivatives of amino sugars, such as sialic acid and N-acetylneuraminic acid are usually
included in this group too. Amino sugars are important components of glycoproteins, of
certain glycosphingolipids (e.g. gangliosides), and of glycosaminoglycans. N-
acetylneuraminic acid is a derivative of sialic acid that is included in the group of amino
sugars. See paper for reactions of synthesis of amino sugars and sialic acid.

Significance of the synthesis of glycoproteins and proteoglycans:

Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached
to polypeptide side chains. In glycoprotein synthesis, before being incorporated into the
oligosaccharide chains, monosaccharides involved in the synthesis are activated by formation
of nucleotide sugars, similarly to the formation of UDP-glucose in the reaction of UTP and
glucose-1-phosphate. The glycosyls of these compounds can be transferred to suitable
acceptors provided that appropriate transferases are available.

44

Glycoproteins are important in the immune system where they act as e.g. antibodies that
interact directly with antigens, molecules of MHC that interact with T-cells as a part of
adaptive immune response. They also form the zona pellucida that covers the oocyte and that
is important in sperm-egg interaction.

Proteoglycans are glycoproteins that are heavily glycosylated. They have a protein core with
one or more covalently attached glycosaminoglycans chains. The chains are linear and
negatively charged under physiological conditions because they contain sulphate and uronic
acid groups. Proteoglycans are major components of connective tissue. The protein
component is synthesized by ribosomes and then translocated into the lumen of the rER.
Glycosylation of the proteoglycans occurs in the Golgi apparatus. The final proteoglycans is
exported to the extracellular matrix through secretory vesicles.

The Uronic acid pathway:

The uronic acid pathway is an alternative oxidative pathway for glucose. It supplies
glucuronic acid and in most animals (NOT human, primates, guinea pigs) also ascorbic
acid. In the liver, the uronic acid pathway catalyzes the conversion of glucose to glucuronic
acid, and pentoses. It is an alternative pathway for glucose that does not lead to the formation
of ATP. See paper for reactions.
The resulting UDP-glucoronate is used in reactions involving its incorporation into
proteoglycans or for reactions with substrates like bilirubin and steroid hormones. In
animals capable of synthesizing ascorbic acid, the glucoronate is reduced to L-gulonate, but
since humans lack the enzyme performing this reaction, gulonolactone oxidase.

31. Metabolism of fructose and galactose, defects:

Metabolism of fructose :

Main sources of fructose obtained from diet is, fruits, honey (60%), sweetened food (salad
dressings, yoghurts, soft drinks). Today the consumption of fructose has increased largely and
we are today consuming about 85-100 grams of fructose /day compared to 16-20 grams of
fructose/day for thousands of years.
Important comparisons between fructose and glucose metabolism is that the intestinal
absorption of glucose is much faster than that of fructose, but the metabolism of fructose (18)
is more rapid than for glucose (43 min). Another aspect is that metabolism of glucose occurs
in most tissues, but metabolism of fructose occurs only in the liver, kidney, and erythrocytes
catalyzed by fructokinase. There is no effect of insulin on fructose, but though on glucose.
Hexokinase has a lower affinity (high K
M
) for fructose than for glucose. See paper for
reactions of fructose metabolism.
Aldolase B is involved in the metabolism of fructose, which has an isoenzyme, aldolase A
that is involved in the glycolysis. Aldolase B cleaves fructose-1-phosphate into
glyceraldehydes and dihydroxyacetone phosphate. Aldolase A cleaves fructose-1,6-
bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
The reason for the very rapid metabolism of fructose is that fructokinase and aldolase B
bypasses the regulated steps and can continuously enter glycolysis, which allows rapid

45

metabolism. Increased amount of fructose leads to the formation of fatty acids that eventually
form triacylglycerols.
Defects in fructose metabolism include lack of fructokinase that lead to accumulation of
fructose in blood and fructosuria. Treatment is fructose-free diet. Another defect might be
lack of aldolase B that leads to accumulation of fructose-1-phosphate that might lead to that
all inorganic phosphates are removed from the cytosol. This condition leads to hypoglycemia
and can be regulated by restricted fructose-and glucose intake.
The production of fructose is possible in the polyol pathway that occurs in many types of
cells, but especially in the liver, kidney, lens and retina. See paper for reactions.
When the glucose concentration in blood is high large amounts of glucose will enter the cells
and it will be metabolized to glucitol by the polyol pathway. Glucitol can however not pass
efficiently through the cell membrane, which then remains trapped inside the cell. Another
defect occurs in tissues deficient of polyol dehydrogenase where glucitol accumulates and
cannot be converted into fructose.

Metabolism of galactose:

Galactose is mainly obtained from the diet by lactose in milk and in other dairy products.
Hydrolysis of lactose in the gut yields galactose and glucose. Metabolism of galactose takes
place in the liver and it is rapidly converted into glucose. See paper for reactions. Galactose
can be used to form, besides glucose, glycolipids, glycoproteins, proteoglycans,
glycosaminoglycans etc.
Defects of galactose degradation include deficiencies of galactokinase (mostly) or
uridyltransferase which lead to an accumulation of galactose and galactose-1-phosphate.
These deficiencies are commonly called galactosemia and if untreated it leads to liver
damages and mental retardation. Galactosemia is very dangerous for newborns and it can lead
to cataract in the lens due to the conversion of galactose into galactitol. Treatment is
restriction of dairy products in the diet.
The biosynthesis of lactate is unique for lactating mammary gland. It involves the enzyme
lactose synthase that consists of a complex of 2 proteins; galactosyl transferase (present in
most tissues), and -lactalbumin (present only in mammary gland during lactation, its
synthesis is stimulated by prolactin).

32. Biosynthesis of fatty acids, control mechanisms:

Biosynthesis of fatty acids:

Synthesis of fatty acids takes place in adipose tissue, liver, lactating mammary gland, and also
in the brain. It is not the reversal of the degenerative process.
Main differences between the synthesis and degradation processes (-oxidation) are that the
synthesis of fatty acids occurs in the cytosol, while the degradation takes place in the matrix
of mitochondria. Intermediates of the fatty acid synthesis are linked to SH-groups of
phosphopantethein of an acyl carrier protein (ACP), not to coenzyme A like in the -
oxidation. Also, in the synthesis the activated donor of 2-carbon units is malonyl-CoA, the
elongation reaction is driven by the release of CO
2
. The reductant in fatty acid synthesis is

46

NADPH, in the degradation the oxidants are NAD
+
and FAD. Required cofactors of lipid
synthesis are: NADPH, ATP, Mn
2+
, biotin, and HCO
3
-
.

The synthesis is initiated by acetyl-CoA being formed in the matrix of mitochondria, mainly
from oxidative decarboxylation of pyruvate (from glucose, amino acids). Since fatty acid
synthesis occurs in the cytosol acetyl-CoA has to be transferred to the cytosol, but since it is
impermeable to the mitochondrial membrane, it is converted into citrate and transported in
this form. This can only occur when there are sufficient amounts of citrate and it is not
necessary in the citric acid cycle. In the fed state, sufficient amounts of glucose are present to
produce acetyl-Co A. When the concentration of ATP is high the degradation of acetyl-CoA
in the citric acid cycle is inhibited. Fatty acids released by the action of catecholamines, and
not by stress, also inhibit fatty acid synthesis.
The transport of acetyl-CoA is catalyzed by citrate lyase:

Citrate + ATP + CoA-SH + H
2
O Acetyl-CoA + ADP + P
i
+ Oxaloacetate

The oxaloacetate released joins the aspartate/malate shuttle.
Acetyl-CoA does not have energy enough to enter the fatty acid synthesis, therefore it is
oxidized and decarboxylated into malonyl-CoA catalyzed by acetyl-CoA carboxylase. This
enzyme is a multienzyme protein and it requires biotin and ATP. The reaction has two steps
(1) carboxylation of biotin involving ATP (2) transfer of the carboxyl group acetyl-CoA so
that malonyl-CoA can be formed. Acetyl-CoA carboxylase is activated by citrate and
inhibited by long acyl-CoA chains (palmitate). It is hormonally activated by insulin and
suppressed by glucagon and adrenalin.

The fatty acid synthase complex consists of two monomers, in both the acyl carrier protein
(ACP) area to which the phosphopantethein arm is attached. This enables two fatty acids
to be synthesized at the same time since the two monomers are cooperating. The fatty acyl
synthase complex consists of seven enzyme activities: acyl/acetyl-CoA transferase, malonyl
transcyclase, condensing enzyme, oxoacyl reductase, hydroxyacyl dehydratase, enoyl
reductase, and palmitoyl thioesterase. The phosphopantethein arm of the enzyme is linked
to a serine residue on the acyl carrier protein ACP, it is also found as one half of the acetyl-
CoA.
Initially, a priming molecule of acetyl-CoA combines with a cysteine-SH group catalyzed by
acetyl translocase. Malonyl-CoA combines with another SH-group located on the 4-
phosphopantetheine of the ACP monomer catalyzed by the malonyl translocase to form
acetyl-malonyl enzyme. The acetyl group attacks the methylene group of the malonyl
residue, catalyzed by 3-oxoacyl synthase and liberates CO
2
forming 3-ketoacyl enzyme
(acetoacetyl enzyme). The 3-ketoacyl group is then reduced, dehydrated and reduced again to
form the corresponding saturated acyl-S-enzyme. Then the ACP is ready for a new round.
See paper for reactions.
After the completion of the first elongating cycle, new malonyl is loaded on the sulfanyl
group phosphopantethein. In the second round of fatty acid synthesis, butyryl unit condenses
with malonyl to form a C
6
-acyl. The cycle continues until a C
16
-acyl unit (palmitoyl) is
formed. Palmitoyl is a good substrate for thioesterase that hydrolyses palmitoyl-PPt to yield
palmitate (16:0). In mammals, palmitate is the major product of FA synthesis. A minor

47

saturated product is stearate (18:0). Further elongation of fatty acids occur by the same
mechanisms, but it is located on the membranes of endoplasmatic reticulum.
NADPH is required in the reductive steps and it is mainly provided by the pentose phosphate
pathway. The NADPH transported by the malic enzyme to the site of synthesis. This reaction
takes place at the same time as the transport of acetyl-CoA in the form of citrate.

Malate + NADP
+
Pyruvate + CO
2
+ NADPH

Synthesis of malonyl-CoA:

7 Acetyl-CoA + 7 CO
2
+ 7 ATP 7 Malonyl-CoA + 7 ADP + 7 P
i
+ 14 H
+

The synthesis of palmitate:

Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 20 H
+


Palmitate + 7 CO
2
+ 14 NADP
+
+ 8 CoA + 6 H
2
O

Regulations:

Control of fatty acid synthesis is regulated by means of reversible phosphorylation of acetyl-
CoA carboxylase. This enzyme is inhibited by phosphorylation by AMP-dependent protein
kinase. De phosphorylation activates the enzyme and this action is insulin-dependent. Local
regulation is provided by citrate that activates the carboxylase, palmitoyl-CoA inhibits acetyl-
CoA carboxylase.

33. The oxidative breakdown of fatty acids (localization, the reaction
sequence, energetic yield, control mechanism):

-Oxidation:

In the -oxidation, fatty acyl-CoA:s are degraded in the mitochondrial matrix by repitition of
four reactions:

1. Dehydrogenation by FAD
2. Hydration
3. Dehydrogenation by NAD
+
4. Thiolysis by CoA

As a result of these reactions, the fatty acyl chain is shortened by 2 carbons, and FADH
2
,
NADH + H
+
and acetyl-CoA is regenerated. The name -oxidation can be derived from that
the carbon is the one to be oxidized. See paper for reactions.

Energetic yield of -oxidation: (respiratory chain)


48

Palmitoyl-CoA + 7 FAD + 7 NAD
+
+ 7 H
2
O + 7 CoA

8 Acetyl-CoA + 7 FADH
2
(14 ATP) + 7 NADH + 7 H
+
(21 ATP)

14 ATP + 21 ATP 2 ATP (activation of palmitate)

8 Acetyl-CoA (citric acid cycle): 8 x 12 ATP = 96 ATP
Net yield: 129 ATP

38 ATP/glucose (aerobic breakdown)
129 ATP/palmitate (aerobic breakdown)
Unsaturated fatty acids and odd-chain fatty acids require additional steps before -
oxidation in order to be oxidized. Examples of unsaturated fatty acids that are included are
oleoyl CoA, and linoleoyl CoA. Odd-chain fatty acids are uncommon, if common; the
product of -oxidation will be propionyl CoA that is carboxylated to methylmalonyl-CoA
and isomerised (B
12
coenzyme) to succinyl-CoA.
-Oxidation is a powerful source of energy; it occurs if the cells require energy and the access
of glucose is not sufficient, e.g. in fasting, stress. Glucagon stimulates the mobilization of
fatty acids and the plasma concentration of fatty acids is increased, which are take up by the
liver and other tissues.

34. The transfer of long-chain fatty acyl-CoA into the mitochondria and
the transfer of acetyl-CoA into the cytosol (control mechanisms):

The transfer of long-chain fatty acids:

Free fatty acids are fatty acids in the non-esterified state. In plasma, longer free fatty acids
are combined with albumin to increase their solubility. Shorter fatty acids exist in free form
as unionized acid or as a fatty anion.
Fatty acids must be activated to an active intermediate before they can be catabolised in the
mitochondrial matrix in the -oxidation. This activation of fatty acids is the only step in the -
oxidation that requires energy, ATP. In the presence of ATP and coenzyme A, the enzyme
acyl-CoA synthetase catalyzes the conversion of a fatty acid into an active fatty acid, acyl-
CoA. Acyl-CoA syntheses are found in the endoplasmatic reticulum, peroxisomes, and on the
outer-and inner mitochondrial membrane.
Long-chain acyl-CoA cannot penetrate the inner mitochondrial membrane, but if it is
combined with carnitine it can. Carnitine palmitoyltransferase-I converts acyl-CoA into
acylcarnitine that is able to penetrate the inner mitochondrial membrane and get access to the
-oxidation. Carnitine acyl-carnitine translocase acts as an inner membrane exchange
transporter, acylcarnitine is transported in, coupled with the transport out of one molecule of
carnitine. Carnitine palmitoyltransferase II located on the inner mitochondrial membrane
reforms acyl-CoA in the matrix and liberates carnitine. See question 19, under anaplerotic
reactions. Short-chain fatty acids can penetrate the inner mitochondrial membrane and does
not require to be transported via carnitine.

35. Ketogenesis - localization, the pathway and the control of it; the

49

utilization of ketone bodies. The circumstances causing ketoacidosis:

Ketogenesis:

Ketogenesis is the process by which ketone bodies (acetone, acetoacetate and -
hydroxybutyrate) are formed from fatty acid breakdown. Since it is formed from the
breakdown of fatty acids it especially occurs when the rate of oxidation is high. Ketone bodies
are formed in the liver mitochondria and released into blood plasma. -Hydroxybutyrate and
acetoacetate are detectable in blood plasma at all times, and the usual ration between
hydroxybutyrate/acetoacetate is about 3-6, which reflects the intramitochondrial
NADH/NAD
+
ratio. There are always traces of ketone bodies in urine since there is no renal
threshold for these two acids, especially during night when no carbohydrates are available.
Ketone bodies are readily metabolized in non-hepatic tissues.
Acetoacetate and -hydroxybutyrate are important in providing energy for peripheral tissues.
Acetone is a waste product that is eliminated by the kidneys or expired; it can be smelled on
the breath. See reactions on paper.
See paper for reactions of ketogenesis. 2 molecules of acetyl-CoA is the initial substrate for
ketogenesis and are derived from the -oxidation. The final outcome of the reaction is acetyl-
CoA that enters the citric acid cycle and acetoacetate that by decarboxylation forms acetone
or that by dehydrogenation forms -hydroxybutyrate. These are then released into blood
plasma and ready for utilization in non-hepatic tissues.
The production of ketone bodies increases at high ratios glucagon/insulin, when fat stores are
mobilized due to starvation, fasting, uncontrolled diabetes mellitus type I etc.
When the body lacks carbohydrates, fat must be broken down into acetyl-CoA in order to get
energy. Acetyl-CoA is not recycles through the citric acid cycle, because citric acid cycle
intermediates (mainly oxaloacetate) have been depleted to feed gluconeogenesis and the
resulting acetyl-CoA are stimulating ketogenesis.
Ketoacidosis:

In extreme production of ketone bodies when the availability of carbohydrates is extremely
limited, ketoacidosis might occur due to that the oxidation of fatty acids is very high and
therefore the ketogenesis is also at a high rate, and since acetoacetate and hydroxybutyrate are
acidic this might decrease the blood pH. Ketogenesis is a proton-producing process that
disturbs the acid-base balance. This leads to an excretion of cations that are electrically
attracted to the two anionic ketone bodies.

36. Desaturation of fatty acids. Polyunsaturated fatty acids (sources and
interconversions, significance):

Desaturation of fatty acids:

Additional double bonds introduced to the already existing monounsaturated fatty acids, are
always separated from each other by a methylene group (except for bacteria). Some
polyunsaturated fatty acids cannot be synthesized by mammals and are nutritionally essential.
Linoleic (n-2) and -linolenic acid (n-3) are the only nutritionally essential fatty acids. In
polyunsaturated fatty acids, double bonds can be introduced at the
4
,
5
,
6
, and
9
, but

50

never beyond the 9
th
position. Only plants can synthesize double bonds beyond the 9
th

position, the also contain 12 and 15 desaturase.
The n-2 and n-3 series are nutritionally essential and serve as precursors of eicosanoids
(leukotrienes and prostanoids). Dietary sources are fish oils and vegetable oils. Linoleic acid
and -linolenic acid are precursors of arachidonic acid (n-6) and eicosapentaenoate (n-3)
from which eicosanoids are formed.
Long-chain fatty acyl-CoA Desaturation takes place in the smooth endoplasmatic reticulum of
liver cells. Desaturases are hydroxylating monooxygenases. The substrate is hydroxylated
and after it, water is eliminated from the hydroxylated product with the formation of a double
bond. The reductant is NADH + H
+
, from which electrons are carried by the flavine enzyme
and the cytochrome b
5
to a desaturase. See paper for reaction of desaturation.

37. Eicosanoids (basic structural types, the initial steps of the synthesis, the
basal features of their function, inhibitors of eicosanoid production as anti-
inflammatory agents):

Eicosanoids:

Eicosanoids are signalling molecules made by oxidation of polyunsaturated 20 carbon
essential fatty acids. They exert control in inflammatory reactions, in the immune system and
ass messengers in the nervous system. Eicosanoids are derived from either omega-3 or
omega-6 essential fatty acids. Their presence in the body will affect the eicosanoid-controlled
functions, e.g. the risk of cardiovascular disease, blood pressure, and arthritis. Eicosanoids
involve 4 types of pharmalocically active substances: prostaglandins, thromboxanes,
leukotrienes and lipoxins.

There are 3 groups of eicosanoids derived from three different C
20
PUFA precursors:
1. Arachidonic (Eicosatetraenoic) 20:4 (5,8,11,14): Originates from linoleic acid
metabolism.

2. Eicosapentaenoic 20:5 (5,8,11,14,17): Originates from linolenic acid metabolism.

3. Dihomo--linolenic (Eicosatrienoic) 20:3 (8,11,14): Originates from linoleic acid metabolism.

See paper for reaction of desaturation and elongation of PUFA:s.

The three groups of eicosanoids are usually obtained from C-2 from membrane
phospholipids by the action of phospholipase A
2
, and it is the substrate for prostanoid
synthesis by the cyclooxygenase pathway. The activity of phospholipase A
2
is regulated by
extracellular mediators like adrenalin and thrombin. Corticosteroids through the action of
lipocortin inhibit phospholipase A
2
.

The cyclooxygenase pathway leads to the synthesis of prostaglandin H, an endoperoxidase,
which is the precursor of cyclic: prostaglandins, prostacyclins and thromboxanes.
Cyclooxygenase (COX) is a membrane-bound enzyme that has cyclooxygenase and
peroxidase activity. It exists in two forms;

51

COX1: constitutive enzyme that is expressed in almost all tissue.
COX2: is inducible, its synthesis is induced by cytokines in inflamed tissue.
COX catalyzes the conversion of arachidonic acid to prostaglandin H, which is the common
precursor of prostanoids of 2-series. Prostanoids mediate partly inflammatory response and by
inhibition anti-inflammatory effects and relief of pain and mitigation of pain. Negative aspects
of inhibition include e.g. decrease in blood platelet aggregation. Inhibitors of cyclooxygenases
include acetylsalicylic acid (aspirin) and paracetamol (ibuprofen). Acetylsalicylic acid
inhibits both COX-1 and COX-2 by acetylation of the active site of the enzyme. Paracetamol
is a reversible inhibitor of COX. Drugs have been developed to only inhibit COX-2.

The lipoxygenase pathway converts precursor acids to acyclic hydroperoxyacids from
which either leukotrienes or lipoxins. Leukotrienes are the most effective eicosanoids.
Eicosanoids are formed in various types of tissue, their site of synthesis depends on gene
expression of enzymes and where these in turn are located; e.g. in blood platelets only
thromboxan synthase is present and in endothelial cells of blood vessels only synthesize
prostacyclins. The catabolism of eicosanoids is rapid and they have a half-life ranging from a
few seconds to a couple of minutes.

38. Biosynthesis of triacylglycerols, the main features of structure and
synthesis of glycerophospholipids:

Biosynthesis of triacylglycerols:

A triacylglycerols is an ester composed of glycerol and three fatty acids. It is the main
component of vegetable oils and animal fats. It is synthesized by esterification of glycerol-3-
phosphate or by dihydroxyacetone phosphate by activated fatty acids acylcoenzymes A.
There are two possible sources of glycerol-3-phosphate:

1. In the liver and small intestine (but not in adipose tissue) is glycerol phosphorylated by
glycerol kinase.
2. In most other tissues glycerol-3-phosphate originates by reduction from dihydroxyacetone
that is an intermediate of glycolysis, by the action of glycerol phosphate dehydrogenase.

See paper for reactions of the biosynthesis of triacylglycerols.

Glycerophospholipids:

Glycerophospholipids are glycerol-based phospholipids that are the major lipids in biological
membranes. Two fatty acids are attached to a glycerol unit that is connected to a phosphate
and a polar differing compound that depends on what type of glycerophospholipids it is.
Possibilities are serine, ethanolamine, choline and inositol. The simplest type of
glycerophospholipids is phosphatidic acid that lacks any group attached to the phosphate. It
occurs only to a small extent in biological membranes and it plays a key role in the synthesis
of other glycerophospholipids.
Biosynthesis of glycerophospholipids is located on the membranes of endoplasmatic

52

reticulum. The component enzymes are integral proteins located on the membrane on the
cytosolic side of the ER. The initial steps of the synthesis are similar to those of
triacylglycerols formation.

There are two mechanisms of adding the head group. In both cases the reaction is driven by
CTP (cytidine triphosphate):

1. Diacylglycerol can accept CDP-activated choline or ethanolamine (synthesis of
phosphatidyl choline (lecithin), phosphatidyl ethanolamine, phosphatidyl serine)

2. Phosphatidate is activated to CDP-diacylglycerol that can accept the head group
(phosphatidyl inositol, cardiolipin)

See paper for reactions of the glycerophospholipids.
Except for being important components in biological membranes, glycerophospholipids are
components if lipoproteins in blood, supply polyunsaturated fatty acids for eicosanoid
synthesis, act in anchoring of some proteins to membranes, serve as components of lung
surfactant etc.
Enzymes catalyzing hydrolysis of glycerophospholipids are called phospholipases.
Phospholipases are present in cell membranes and lysosomes and they hydrolyze the substrate
at specific ester bonds. There are phospholipases A
1
, A
2
, C, D.

39. Degradation of triacylglycerols. Lipases-their function and types:

Degradation of triacylglycerols:

Triacylglycerols are very hydrophobic compounds that are very insoluble in water. In the
presence of tensides they are emulsified into micelles. Milk is an emulsion of triacylglycerols
in water. Triacylglycerols belong to the simple lipid group and it is the main storage form of
fatty acids in the human body. Triacylglycerols play an important part in metabolism as
sources of energy and transporters of dietary fat.
Lipolysis is the metabolic breakdown of triacylglycerols into free fatty acids within the cell.
Triglycerides are transported through the blood to the liver and adipose tissue by
lipoproteins such as chylomicrons. Triglycerides present in the chylomicrons undergo
lipolysis by lipases. Lipases are enzymes that catalyze hydrolysis of ester bonds of
triacylglycerols and by these releasing free fatty acids. There are two major types of lipases:

- Extracellular lipases:

Pancreatic lipase: Secreted into the duodenum.
Lipoprotein lipase: Secreted on the surface of the endothelium lining the capillaries.

- Intracellular lipases:

Hormone-sensitive lipase of adipocytes: Mobilizing fat stores

53

Lysosomal lipase

Hormone-sensitive lipases of adipocytes are responsible of mobilization of fat energy stores.
It is receptive to hormones and it responds to glucagon that is raised during low blood
glucose and that stimulates lipolysis of triacylglycerols. Noradrenalin and adrenalin are
secreted during stress and these stimulate also lipolysis by phosphorylation of lipase and thus
activate it. Insulin, though, have the opposite effect and activates dephosphorylation of
hormone-sensitive lipase (inactivation), which suppresses lipolysis and promotes synthesis of
triacylglycerols.

40. Biosynthesis of cholesterol (the most important reactions and stages,
regulation), excretion of cholesterol and the cholesterol balance in the body:

Biosynthesis of cholesterol:

Cholesterol is a component of cell membranes that is transported in blood plasma of animals.
It provides rigidity in biological membranes and it is also used in synthesis of steroids, bile
acids and vitamin D
3
. Cholesterol is produced in most tissues, but mainly in liver,
reproductive tissues, adrenal cortex and red blood cells. The actual synthesis is located in the
cell plasma; some enzymes are located on the ER.

Cholesterol is synthesized from acetyl-CoA and is performed in three main steps, see paper
for detailed reactions:

1. First phase: Synthesis of 3-HMG-CoA from acetyl-CoA.

2. Second phase: Formation of mevalonic acid from 3-HMG-CoA.

The synthesis of mevalonic acid determines the overall rate of the cholesterol synthesis. The
3-HMG-CoA reductase that catalyzes this double reduction of the carboxylic group into a
primary alcohol is situated on the membrane of the ER; it serves as a major control of the
synthesis and is inhibited by several drugs. The metabolic controls of this reductase involve
control of enzyme synthesis by sterol levels (affection of gene transcription by transcription
factor SREBP, released at low levels of cholesterol), control of enzyme proteolysis by the
sterol level, control of enzyme activity by covalent modifications (phosphorylation) and
inhibition by competitive drugs statins (lovastatin, pravastatin, cerivastatin).

Proteolysis of HMG-CoA reductase is stimulated by high amounts of cholesterol,
mevalonate and farnesol. High levels of sterols stimulated proteolysis.
When the reductase is phosphorylated it is inactive which is catalyzed by AMP-dependent
protein kinase. Protein kinase in turn is stimulated by glucagon, intracellular sterols
(cholesterol, bile acids), and glucocorticoids.
The active form is when the enzyme is in the dephosphorylated state catalyzed by
phosphatase. It is stimulated by insulin and thyroidal hormones.
Statins inhibit 3-HMG-reductase in the liver and act as competitive inhibitors since their
structure resembles HMG-CoA. Examples of statins are lovastatins, pravastatins, simvastatins

54

etc.

3. Third phase: Formation of 5-carbon units.

Dimethylallyl diphosphate and isopentenyl diphosphate are the two five-carbon units formed.
From these other compounds important in syntheses are formed. See paper.
Squalene with 30 carbons, formed from 2 molecules of farnesyl diphosphate, is the substrate
for cholesterol synthesis. It involves 19 steps in the ER and involves mainly:
- Cyclisation
- Shortening carbon chain from 30 to 27 carbons
- Movement of double bonds
- Reduction of double bond

The cholesterol balance in the body:

The cholesterol balance is regulated by that cell cholesterol increases due to uptake of
cholesterol-containing lipoproteins by receptors, e.g. LDL receptor or uptake of free
cholesterol from hydrolysis of cholesteryl esters.
A decrease of cholesterol is due to a efflux (= utstrmning) of cholesterol from the membrane
to HDL that in turn can be used for esterification of cholesterol via ACAT (acyl-CoA-
cholesterol acyl-transferase) and for synthesis of other steroids, such as hormones, or bile
acids in the liver.
The lipoproteins that carry the cholesterol have different cell signals that direct them to
different tissue; they can be classified into 5 groups with increasing density; chylomicrons,
VLDL (very low density lipoprotein), IDL (intermediate density lipoprotein), LDL (low
density lipoprotein), and HDL (high density lipoprotein). The more cholesterol and less
protein is has the less dense it is. The cholesterol carried within the lipoproteins is identical,
however, sometimes it is carried as cholesterol esters. Each type of lipoprotein has
apolipoproteins that serve as receptors and directs where the cholesterol is heading.
The receptors of LDL transport the cholesterol into peripheral tissues where it becomes a
component of the cell membrane which contributes to rigidity of it which is thought to
contribute to arteriosclerosis. VLDL is converted into LDL after its triacylglycerols have been
utilized in the liver, then the LDL heads to the peripheral tissues. The receptors of HDL
transport the cholesterol back to the liver where it can contribute in production of sterols, e.g.
hormones. Recommended concentration of cholesterol in blood is that it should be less than 5
mmol/l.
Decomposition of cholesterol is only possible in the liver and it can be eliminated either by
conversion into bile acids and excreted by them, or by excretion of free cholesterol in bile.
Small amount of cholesterol is excreted as earwax, and a small amount is used for synthesis of
steroids and vitamin D
3
.

41. Synthesis of bile acids - localization, main steps of the synthesis,
secretion and elimination from the body:


55

The primary bile acids are synthesized in the liver from cholesterol. See paper for reactions
of the synthesis of primary bile acids. Primary bile acids include cholic acid (found in the
largest amount) and chenodeoxycholic acid. The 7-hydroxylation of cholesterol is the first
and principal regulatory step of the biosynthesis of bile acids and it is catalyzed by
cholesterol 7-hydroxylase, a typical monooxygenase that requires NADPH, oxygen and
P450. Subsequent hydroxylation steps are also catalyzed by monooxygenases. The
monooxygenase reactions are located in the ER of the liver. The biosynthesis of bile acids
divides early into one sub-pathway producing cholate (-hydroxyl groups at positions 3, 7
and 12) and chenodeoxycholate (-hydroxyl groups at positions 3 and7). These are the
primary bile acids. Three carbons from the side chain are removed by an oxidation reaction.
These primary bile acids are conjugated with either taurine or glycine in the ER of the liver
before they enter the bile and their conjugated form is assumed to be in the bile salt form. The
conjugation of primary bile acids increases the detergent efficiency of bile in the intestine in
emulsifying fat. Other functions of bile are to eliminate cholesterol from the body etc.

Secondary bile acids include lithocholic acid and deoxycholic acid and are formed in the
intestine when primary bile acids are subjected to further changes of the activity of the
intestinal bacteria. These changes include deconjugation and 7-dehydroxylation which
produce the secondary bile acids.

The enterohepatic circulation:

Although products of fat digestion, including cholesterol, are absorbed in the first meter of the
small intestine, the primary and secondary bile acids are absorbed almost exclusively in the
ileum and 98-99% are returned to the liver again by the hepatic portal circulation. This is
known as the enterohepatic circulation. However, lithocholic acid, because of its insolubility,
is not absorbed to any significant extent. Only a small fraction of the bile acids escape the
absorption and are excreted in the feces.

42. Nutritionally essential amino acids. Biosynthesis of nonessential amino
acids (Asp, Glu, Ser, Pro, Cys, Tyr):

Essential amino acids:

Essential amino acids in human diet are; isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, arginine, histidine and valine. Histidine and
arginine are only essential in children. 30% of methionine can be synthesized from cysteine
and 50% of phenylalanine can supply tyrosine.

Biosynthesis of non-essential amino acids:

Aspartate: Aspartate is formed from transamination of oxaloacetate.

Glutamate: Glutamate is formed by reductive amidation of -ketoglutarate (2-oxoglutarate)
catalyzed by glutamate dehydrogenase. This reaction constitutes the first step in synthesis of
the glutamate family. This reaction requires either NADH or NADPH as cofactor. Also,
ammonium is supplied from the urea synthesis which is converted into water.


56

Serine: Serine is formed from two subsequent reactions. Oxidation of the -hydroxyl group
of the glycolytic intermediate 3-phosphoglycerate by 3-phosphoglycerate dehydrogenase
converts it into 3-phosphohydroxypyruvate. Transamination and subsequent
dephosphorylation forms serine.

Proline: Proline is synthesized from glutamate in a series of four reactions that require
NADPH and ATP. The biosynthesis reactions are very similar to the catabolism reactions.

Cysteine: Cysteine is formed from methionine, which is nutritionally essential. Methionine is
converted into homocysteine, homocysteine and serine form cystathione, whose hydrolysis
form cysteine and homoserine.

Tyrosine: Phenylalanine hydroxylase converts phenylalanine to tyrosine. If the diet contains
adequate amounts of phenylalanine, tyrosine is non-essential. Since the phenylalanine
hydroxylase reaction is irreversible phenylalanine cannot be supplied by tyrosine. One atom
of oxygen is incorporated into the structure of phenylalanine and reduces the other oxygen
atom into water. The reducing power is provided by tetrahydrobiopterin.

43. Intracellular degradation of proteins (proteasomes, lyzosomes):

Degradation of proteins:

Proteolysis is the degradation of proteins by cellular enzymes called proteases or by
intramolecular digestion. The main sources of amino acids are by proteolysis of dietary
proteins, proteolysis by tissue proteins and synthesis of non-essential amino acids. Main
utilizations of amino acids is in the synthesis of blood plasma and tissue proteins, synthesis of
low-molecular nitrogen compounds (e.g. hormones), and deamination and utilization of the
amino acid carbon skeletons. Proteins that are degraded can be divided into two groups:

- Exogenous proteins: These proteins are degraded in the gastrointestinal tract, in the
stomach by pepsin, and in the intestine by pancreatic proteases (trypsin,
chymotrypsin etc.)

- Endogenous proteins: These proteins are degraded by intracellular proteases by two
systems (1) lysosomes, and (2) ubiquitin-proteasomes.

Lysosomes:

The degradation of proteins in lysosomes is a non-specific reaction that does not require ATP.
Proteolytic enzymes of lysosomes are called acid hydrolases that have a pH optimum of
about 4,5. The Lysosomal enzymes are produced in the cytoplasm and endoplasmatic
reticulum (e.g. lipase, collagenase, elastase). Proteins that are degraded are extracellular
proteins, membrane proteins, and long-lived intracellular proteins. In the case of extracellular
glycoproteins, sialic acid on the terminal position of the oligosaccharide is removed and thus
gives rise to asialoglycoproteins that are degraded by liver lysosomes.

Proteasomes:

57


In contrast to lysosomes, proteins degraded by the action of proteases are regulatory proteins
with short half-life and of abnormal and misfolded proteins. Proteolysis by proteasomes takes
place in the cytoplasm, is ATP-demanding and requires ubiquitin, a protein present in all
eukaryotic cells. For a protein to be degraded by the proteasome it has to be market by
ubiquitin. The mechanism of attachment of ubiquitin to a target protein involves three
enzymes; an activating enzyme, a conjugating enzyme, and a ligase. The binding of
ubiquitin to the activating enzyme requires ATP. Ubiquitin binds by its C-terminus to SH-
groups of proteins that are to be degraded.
Once one molecule of ubiquitin is attached to the protein, several other molecules of ubiquitin
bind to the protein, which results in polyubiquitination. The polyubiquinated protein then
enters the proteasome; the ubiquitin is released by deubiquitination by deubiquinating
enzymes and it can be used for targeting of another protein.
The proteasome is located in the cytoplasm and has a large cylindrical structure composed of
28 polypeptides. It consists of a hollow core and two caps located on the ends. The caps
regulate the entry of proteins into the destruction chamber by hydrolysis of ATP. Inside the
barrel, specific proteases hydrolyze the target protein into short peptides. Bortezomib act as
an inhibitor to the proteasome by its boron atom that binds to the active site.

44. Deamination of amino acids and transamination (deamination types,
reaction course, coenzymes, consequence of reactions in removal of amino
groups from amino acids):

Deamination & transamination of amino acids:

See paper for typical transamination reaction. Transamination is a reaction between an amino
acid and an -keto acid where the amino group is transferred from the amino acid to the -
keto acid (2-oxo acid). This action results in that the -keto acid is transformed into a
corresponding amino acid and that the amino acid is converted into a corresponding -keto
acid. Transamination is accomplished by enzymes called aminotransferases that require the
cofactor pyridoxal phosphate. The presence of aminotransferases is important in the
production of amino acids that are not present in the diet. Serine and threonine are the only
amino acids that dont undergo transamination.
Transamination has two phases; in the first phase pyridoxal phosphate, by its aldehydes
group, reacts with the nitrogen group of the amino acid and together give rise to a Shiff base.
The Shiff base is then isomerised to an imino acid. By hydration, the Shiff base is converted
into an oxo acid.
During the second phase of the transamination, the pyridoxal amine reacts with the ketone
carbon that by dehydration forms an imino acid (Shiff base). By isomerisation and then
hydration pyridoxal phosphate and glutamate are formed.
In transaminations, nitrogen of most amino acids is concentrated in glutamate. Glutamate then
undergoes dehydrogenation and deamination and releases free ammonia NH
3
. The
transamination reactions leading to glutamate occurs in the cytosol. Glutamate is then
transported into mitochondria where it is dehydrogenated and hydrolyzed into ammonia and
2-oxoglutarate. This conversion of -amino nitrogen into free ammonia is often termed
transdeamination. The reaction is reversible and can also function in the purpose of

58

synthesizing amino acids. Liver glutamate dehydrogenase is allosterically inhibited by ATP,
GTP and NADH, and activated by ADP.

45. Detoxification of ammonia (the ureosynthetic cycle, glutamine,
glutamate):

Detoxification of ammonia:

There are two main sources of ammonia NH
3
in the body;

1. Deamination of glutamate (GMD reaction) in tissues.
2. Bacterial putrefaction of proteins in the large intestine: produces nitrogen catabolites
(e.g. biogenic amines + ammonia), ammonia diffuses freely into portal blood since the portal
blood has high concentration of ammonium NH
4
+
, which is then eliminated by the liver.

Other sources of ammonia include deamination of adenine, oxidative deamination of some
amino acids that lead to the formation of H
2
O
2
, oxidative deamination of terminal NH
2
of
lysine, dehydration deamination of serine, oxidative deamination of biogenic amines,
synthesis of heme, hydrolysis of amide group in glutamate etc.

Ways to decrease the ammonia production in the body include e.g. a low-protein diet,
alteration of the colon microflora by ingestion of probiotics that contain bacteria that
stimulate the fermentive processes in the large intestine instead of the putrefactive ones.
Sources of probiotics are in yoghurt, kefir milk etc. By ingestion of probiotics, that is non-
digestible polysaccharides, the growth probiotics is stimulated in the colon.

There are three ways of ammonia detoxification:

1. Formation of glutamine from glutamate + NH
3
:

This reaction is catalyzed by glutamine synthase and requires 1 ATP. It occurs in the
cytoplasm of mainly liver and brain. Glutamine is also used for detoxification of ammonia
and it is also used in the synthesis of glutamate that can be used in the synthesis of other
amino acids. It is used as a transporter of ammonia in a non-toxic form. Besides being a
source of ammonia for urea synthesis it is also used as a source of nitrogen for synthesis of
purine, pyrimidine, NAD
+
and amino sugars.

2. Hydrogenation and deamination of 2-oxoglutarate to form glutamate:

Glutamate dehydrogenase catalyzes the reversible reaction that requires 1 NADPH + H
+
. It
occurs in the mitochondria of the brain.

3. The urea cycle:

The urea cycle consists of 5 reactions where reactions 1 and 2 occurs in the mitochondria and
reaction number 3-5 occurs in the cytosol. See paper for reactions. The urea synthesis requires
3ATP.

59

CO
2
+ NH
4
+
+ aspartate urea + fumarate + H
2
O + 2 H
+


As can be seen from the reaction, the urea synthesis is a proton-productive reaction leading to
decreased pH. Urea itself is a polar compound that is soluble in water and diffuses easily
through cell membranes through hydrophilic pores. It is synthesized in the liver and its
excretion depends on the amount of ingested proteins, but on average it is 20-35 g/day. Urea
also contributes to plasma osmolality. Normal concentration of urea in blood is about 2-8
mmol/l. Increased concentrations indicate renal failure and increased protein catabolism, e.g.
due to sepsis, burns etc. Decreased amounts indicate liver failure or lack of proteins in the
diet. In kidneys, ammonia is released from glutamine and glutamate. Ammonium cation is
excreted by urine.

46. Glucogenic and ketogenic amino acids ("families" according to the
resulting amphibolic intermediates, reversible interconversions of amino
acids, essential amino acids):

Glucogenic amino acids:

A glucogenic amino acid is an amino acid that can be converted into glucose by the
gluconeogenesis. The production of glucose from glucogenic amino acids involves some
amino acids that are converted into -keto acids through pyruvate or as citric acid cycle
intermediates which are then converted into glucose. These processed occur in the liver. It
occurs especially during fasting or starvation. Glucogenic amino acids are: glycine, serine,
valine, histidine, arginine, cysteine, prolin, alanine, glutamate, glutamine, aspartate,
asparagine, and methionine.

Ketogenic amino acids:

Ketogenic amino acids can be converted into ketone bodies through ketogenesis. Ketogenic
amino acids are unable to form glucose. In humans, ketogenic amino acids include leucine
and lysine. They are converted into acetyl-CoA and acetoacetate.

Some amino acids can both be converted into glucose and ketone bodies and are referred to as
mixed amino acids which include threonine, isoleucine, phenylalanine, tryptophan, and
tyrosine.

Pyruvate: Seine, glycine, threonine, alanine, cysteine, tryptophan
Acetyl-CoA: Isoleucine, leucine, lysine, threonine
Acetoacetate: Leucine, lysine, phenylalanine, tryptophan, tyrosine
2-oxoglutarate: Arginine, glutamate, glutamine, histidine, prolin
Succinyl-CoA: Isoleucine, valine, methionine
Fumarate: Phenylalanine, tyrosine
Oxaloacetate: Aspartate, asparagine

Interconversions of amino acids:

60


See paper for more detailed reactions.

Serine Glycine

Proline Pyrrolidine-5-carboxylate Glutamate-5-semialdehyde Glutamate

Glutamate + NH
3
Glutamine

Phenylalanine Tyrosine

47. Metabolism of dicarboxylic amino acids:

Dicarboxylic amino acids include glutamate and aspartate that are acidic and glucogenic
amino acids.
Glutamate is a non-essential amino acid, see paper for reactions; transamination,
dehydrogenation deamination, decarboxylation. Glutamate is produced in most
transaminations of amino acids by means of 2-oxoglutarate. In transamination, aspartate is
formed which is then used in the urea cycle where it gives rise to one of the nitrogen in urea.
Transamination of glutamate is a reversible process, as well as dehydrogenation deamination.
Dehydrogenation deamination of glutamate is the main source of ammonia in tissues.
Glutamate is readily produces from histidine and proline.

Aspartate produces oxaloacetate by its transamination and -alanine by its decarboxylation. In
the urea cycle, aspartate donates nitrogen, as well as in pyrimidine synthesis. Aspartate is a
non-essential amino acid. See paper for reactions.


48. Metabolites and specialized products of proline, histidine, and
tryptophan significant in metabolism:

Proline:

Proline is converted into glutamate by a non-transamination reaction. See paper. Also, when
2-oxoglutarate is decarboxylated into succinate, proline is hydroxylated into 4-
hydroxyproline. 4-Hydroxyproline in turn is catabolised into pyruvate. Proline is a non-
essential amino acid.

Histidine:

Deamination of histidine provides urocanic acid, or urocanate. Urocanate is present in the skin
where it protects it from harmful UV-radiation. Urocanic acid then forms FIGLU by
hydration and oxidative splitting of the imidazole ring. In the presence of tetrahydrofolate the
formimino group of FIGLU is transferred to the tetrahydrofolate and glutamate is formed. If
tetrahydrofolate is deficient or totally absent the formation of glutamate is not possible and
this can be used in the detection of tetrahydrofolate deficiency.

61

Decarboxylation of histidine provides histamine that stimulates the secretion of HCl in the
stomach, is released in allergic reactions and trigger inflammatory responses. Histidine itself
is responsible for the buffering action of proteins, and is an essential amino acid in childhood.
It does not undergo transamination, but is desaturated and deaminated. Since it is converted
into glutamate it is a glucogenic amino acid.

Tryptophan:

Decarboxylation of tryptophan leads to the formation of tryptamine that functions as a
neurotransmitter in mammals. Tryptophan is also the substrate for the formation of melatonin
and serotonin. Serotonin is known to contribute to the state of well-being. Melatonin regulates
the sleep-wake cycle and it is also a hormone secreted in darkness. Tryptophan itself is an
essential amino acid that does not undergo transamination, amino group leaves as alanine.

49. Conversions of arginine, utilization of the guanidine part (biosynthesis
of creatine, nitroxide formation):

Arginine is an essential amino acid in children and it is the most basic amino acid that does
not undergo transamination. Hydrolysis of arginine leads to the formation of urea and
ornithine. Ornithine can then be converted into glutamate. See papers for reactions of the
biosynthesis of creatine and nitroxide formation. Since arginine releases NO it acts as a
vasodilator. Arginine does not undergo transamination, but is converted into ornithine and
urea.

50. Conversions of glycine and serine, the utilization in anabolic pathways
(one-carbon units, aminolevulinate, purine, creatine, conjugation to
aromatic acids):

Serine:

Serine forms pyruvate and ammonia NH
3
by dehydration and deamination, thus it is a
glucogenic amino acid. Serine can be converted into glycine. Transamination of serine forms
eventually glucose and thus it is a glucogenic amino acid. Decarboxylation and methylation of
serine leads to the formation of choline that is a part of quaternary ammonium salts that are
found e.g. in lipids making up cell membranes. Oxidation of choline leads to the formation of
betaine that is an osmolyte. Serine is a non-essential amino acid that is a component of
glycerophospholipids.

Glycine:

Catabolism of glycine leads to complete oxidation into ammonia and carbon dioxide,
methylene group is transferred to tetrahydrofolate. Oxidative deamination leads to the
formation of oxalate. Other source of oxalate are from the diet (rhubarb, spinach, mangold
etc.) and also from catabolism of vitamin C. Glycine is formed from serine and it is used in
formation of porphyrines (aminolevulinic acid that is used in the synthesis of heme), purine
bases, creatine, glutathione etc. It is also a conjugation agent for bile acids.


62

51. Metabolism of sulphur-containing amino acids. Selenocysteine:

Sulphur-containing amino acids include cysteine and methionine.

Methionine:

Methionine is an essential amino acid that is metabolized into cysteine, which makes cysteine
non-essential. The final catabolite of methionine is succinyl-CoA that is a part of the citric
acid cycle, which means that methionine is a glucogenic amino acid. Methionine is a
methylation agent that gives homocysteine as a side product. Methionine joins ADP and
forms S-adenosylmethionine that can methylate a substrate. After giving off the methyl group
it forms S-adenohomocysteine that is then converted into homocysteine that is a harmful
compound. Its mechanism is yet not fully understood, but it supports the formation of oxygen
radicals and increases the LDL lipid peroxidation. Elevated levels of homocysteine in blood
increases the risk of cardiovascular disease. To eliminate homocysteine three vitamins are
needed; folate, cobalamin and pyridoxine.

Cysteine:

Cysteine is made from methionine, see paper for reaction. It leads to the formation of
pyruvate and thus it is a glucogenic amino acid. Cysteine provides taurine that can be used in
conjugations of bile acids. Cysteine is also a part of the antioxidant glutathione. It is also
responsible for the formation of sulphite, sulphite via sulphite oxidase, catalyzes the
formation of sulphate that is used in PAPS and via blood plasma excreted through the urine.

Selenocysteine is an amino acid that is present in several enzymes e.g. glutathione peroxidase
and glycine reductase (antioxidant activity). The structure of selenocysteine is similar to that
of cysteine, but instead of a sulphur atom an atom of selenium has replaced it. Proteins that
contain one or more selenocysteine residues are called selenoproteins. It is not encoded by a
special UGA stop codon and not like other amino acids.

52. Glutathione - structure, functions (reducing effect, conjugations with
GSH):

Glutathione:

Glutathione is a tripeptide consisting of the amino acids cysteine, glutamate and glycine.
These amino acids are not essential and thus glutathione is a compound that the body can
synthesize itself. The SH-group of cysteine serves as a proton donor and has reducing effects
that can e.g. reduce reactive oxygen species. Glutathione exists in one oxidized form and one
reduced form. Besides its function in reducing reactive oxygen species and act as an
antioxidant, it is also important in maintaining the reduced states of exogenous antioxidants
like e.g. vitamin C and E. In conjugation with hydrogen peroxide following reaction occurs:

H-O-O-H + 2 GSH G-S-S-G + 2 H
2
O

Glutathione is regenerated by the oxidation of NADPH + H
+
into NADP
+
.

63


53. Catabolism of tyrosine; metabolic disorders of tyrosine catabolism:

Catabolism of tyrosine:

Phenylalanine is an essential amino acid that forms tyrosine if it is present in sufficient
amounts. Phenylalanine is hydroxylated into tyrosine by the action of tetrahydrobiopterine
cofactor. Tyrosine provides fumarate that is an intermediate in the citric acid cycle, and it also
provides acetoacetate that is an intermediate in the ketone body formation. This makes
tyrosine a mixed amino acid, both glucogenic and ketogenic. Tyrosine can also form
hormones, e.g. by hydroxylation and addition of thyreoglobulin it forms thyroxine that is
secreted by the thyroid gland. It also forms catecholamines and melanin that is a pigment
present in skin and hair. Metabolic disorders of tyrosine catabolism include:

- Tyrosinemias:
There are two types of Tyrosinemias; type I and type II. Tyrosinemia type I the
enzyme fumarylacetoacetate hydrolase, that forms fumarate and acetoacetate from
fumarylacetoacetate, is deficient or absent that leads to an accumulation of
fumarylacetoacetate. Therapy involves a diet low in tyrosine and phenylalanine. This
disorder leads to death due to liver failure if it is untreated. In tyrosinemia type II the
enzyme tyrosine aminotransferase, that converts tyrosine into
hydroxyphenylpyruvate, is deficient. Therapy involves a diet low in protein.

- Alkaptonuria:

In this disorder the enzyme homogentisate oxidase has reduced activity. The urine
darkens on exposure to air due to oxidation of homogentisate to benzoquinone acetate.
Excessive amount of homogentisate causes damage to cartilage and heart valves as
well as participating in the formation of kidney stone. Alkaptonuria is an inherited
recessive disorder.

54. Hydroxylation of phenylalanine, tyrosine, and tryptophan (coenzyme,
phenylketonuria, DOPA, serotonin):

Phenylalanine:

Hydroxylation of phenylalanine gives tyrosine accompanied by the co-reductant
tetrahydrobiopterine. See paper for reaction. Phenylketonuria is a disorder that can occur in
different variants. For example, phenylalanine hydroxylase might be deficient or
dihydrobiopterine reductase. Consequences of phenylketonuria involve e.g. mental
retardation. Treatment is a diet low in phenylalanine.

Tyrosine:

Hydroxylation of tyrosine via tetrahydrobiopterine leads to the formation of DOPA. Further
decarboxylation of DOPA leads to the formation of dopamine that is a catecholamine

64

neurotransmitter.

Tryptophan:

Hydroxylation of tryptophan leads to the formation of 5-hydroxytryptophan that also requires
the coenzyme tetrahydrobiopterine. 5-hydroxytryptophan is a precursor of serotonin that is
formed by its decarboxylation. By N-acetylation and O-methylation, melatonin is formed
from serotonin. See paper for reactions.

55. Significance and the basic features of the branched-chain amino acids
catabolism:

Branched amino acids include: leucine, isoleucine and valine. All of these amino acids are
essential. The first two steps involve transamination and decarboxylation. The original
branched amino acid is transaminated into a 2-oxo acid and then oxidatively decarboxylated
into a branched acyl-CoA. Following this, the acyl-CoA is dehydrogenated by FAD and
converted into a branched unsaturated acyl-CoA. There are different fates for the three
branched amino acids. Leucine is converted into acetyl-CoA (ketogenic), isoleucine is
converted into acetyl-CoA and succinyl-CoA (ketogenic + glucogenic), and valine is
converted into succinyl-CoA (glucogenic). The first three reactions are the same for all the
branched amino acids; transamination, oxidative decarboxylation, and dehydrogenation.
Branched chain amino acids are mostly utilized in the brain and muscle, since the liver lack
the amino transferase.

56. Decarboxylation of amino acids (coenzyme, some physiologically
important reaction products and significance of them):

Decarboxylation of amino acids lead to the formation of biogenic amino acids that are
biologically important compounds. Decarboxylation is accomplished in the presence of
pyridoxal phosphate as a cofactor. Examples of products of decarboxylation are histamine
from histidine (in allergic reactions, inflammatory response, neurotransmitter), tyramine from
tyrosine (associated with headache and high blood pressure), -alanine from aspartate
(occurring in pantothenic acid).

57. Biosynthesis of catecholamines:

Catecholamines are released during stress from the adrenal glands as a response to fight or
flight. Catecholamines are derived from tyrosine. Most abundant catecholamines are
epinephrine (adrenalin), norepinephrine (noradrenaline), and dopamine that is formed
from DOPA. DOPA can then be further decarboxylated into dopamine, then dopamine can be
hydroxylated into noradrenaline, further on adrenaline is formed from N-methylation.
Decarboxylation of glutamate provides GABA. See paper for detailed reaction.

58. Biosynthesis of creatine, function in muscles, conversion and excretion:

Arginine and glycine are joined together to form guanoacetate and ornithine.
Guanidinoacetate is methylated on the first nitrogen to form creatine. Creatine can then be

65

further phosphorylated by ATP into phosphocreatine. Creatine is used in muscle in that it
forms creatine phosphate that is a phosphagen and acts as a reserve of inorganic phosphate for
fast and rapid ATP formation, faster than anaerobic glycolysis. Creatine is degraded by non-
enzymatic cyclization dehydration into creatinine. Creatinine is excreted by the kidneys and it
is to a very small extent re-absorbed. See paper for all reactions.

59. Biosynthesis of haem. Porphyrias:

Synthesis of haem:

Heme is a prosthetic group in many proteins e.g. hemoglobin, myoglobin, peroxidase, catalase
and cytochromes. Hemoglobin is mainly synthesized in the bone marrow, cytochromes and
other hemoproteins are synthesized in the liver. Heme consists of a porphyrin ring
coordinated with an iron cation. The initial for synthesis is succinyl-CoA that is an
intermediate in the citric acid cycle. The source of nitrogen for porphyrin synthesis is
provided from glycine. The synthesis takes place in mitochondria and in the cytosol. See
paper for reactions.
Glycine and succinyl-CoA are condensed accompanied by the removal of CoA-SH catalyzes
by ALA-synthase that requires pyridoxal phosphate. 2-amino-3-oxoadipate is formed which
is then further decarboxylated into -aminolevulinate. The regulation of this first phase is by
ALA-synthase. It is allosterically inhibited by heme and stimulated by some drugs, e.g.
barbiturates. Until now all reactions have taken place in the mitochondria.
In the cytosol, two molecules of -aminolevulinate condense and are then dehydrated into
porphobilinogen. Porphobilinogen is a substituted pyrrole that forms either uroporphyrinogen
I or III, where uroporphyrinogen III is the major product. Uroporphyrinogen is a condensed
system of four porphobilinogen systems that are deaminated when forming uroporphyrinogen.
Uroporphyrinogen undergoes decarboxylation of four carboxyl groups which forms the
methyl groups. The compound formed from this is coproporphyrinogen III. Two propionic
acids form vinyl groups of coproporphyrinogen III which forms protoporphyrinogen IX.
Heme is a chelate of protoporphyrinogen IX and Fe
2+
.

Porphyrias:

Porphyrias is a collective name for inherited disorders caused by one of the heme synthesizing
enzymes. Primary porphyrias are caused by e.g. defective enzyme in the heme production,
overproduction or accumulation of intermediates etc. Secondary porphyrias are when the
enzyme is inactivated as a consequence of disease or poisoning. Porphyias colour the
excrement and urine with a purple discoloration.

60. The basic steps in purine and pyrimidine nucleotide biosynthesis (the
compounds donating the nitrogen and carbon atoms of the heterocyclic
rings) and the regulation:

Biosynthesis of purine and pyrimidine nucleotide biosynthesis:

The synthesis of ribonucleotides, deoxyribonucleotides and their phosphates is crucial to all

66

cells because it is necessary for the cell to replicate and nucleotides are poorly absorbed from
the diet and thus has to be produced endogenously. Nucleotides participate not only in the
replication of DNA, they are also a part of the energy metabolism, are main donors and
acceptors of phosphoryl groups, they also function in the activation of certain compounds, e.g.
in UDP-glucose in the synthesis of glycogen.
The conversion of ribonucleoside diphosphate into 2-deoxyribonucleotide diphosphate is by
the oxidation of thioredoxin. When thioredoxin is reduced again this requires NADPH.
Intracellular mechanisms sense and regulate the pool size of the nucleotide triphosphates,
which rise during growth or tissue regeneration when cells are rapidly dividing. Purines and
pyrimidines are synthesized in slightly different ways including that in pyrimidines the
pyrimidine ring is synthesized before the ribose is synthesized, in purines, phosphoribosyl
diphosphate is the starting compound in the synthesis and the ribose is built step-by step by
PRDP as a primer with one-carbon fragments. Phosphoribosyl diphosphate is synthesized by
the kinase enzyme PRDP-synthetase where ribose-5-phosphate forms phosphoribosyl
diphosphate by the hydrolysis of ATP into AMP.

Biosynthesis of pyrimidines: Initial substrate for pyrimidine synthesis is glutamine that by
participation of CO
2
and ATP forms carbamoyl phosphate catalyzed by carbamoyl phosphate
synthase II that is the main site for regulation. It is inhibited by UTP and activated by PRDP.
This reaction takes place in the cytoplasm. By a series of reactions UMP is formed that by
phosphorylation of ATP forms UDP. UDP can then have two alternative pathways depending
on is CTP or TMP is desired to be formed. In the case of CTP, UDP is further phosphorylated
into UTP that then forms CTP by the conversion of glutamine into glutamate and the addition
of ATP. This reaction is catalyzed by CTP synthase. In the case of TMP, UDP is converted
into dUDP by the participation of NADPH and catalysis by ribonucleotide reductase. dUDP
is then converted into dTMP by the action of thymidylate synthase and the participation of
tetrahydrofolate that performs methylation.
Pyrimidines can also be formed from the salvage pathway that is an alternative synthetic
pathway for nucleotides. Nucleotides are synthesized from intermediates in the dergradative
pathway of nucleotides. Relatively non-specific pyrimidine nucleoside phosphorylase
converts pyrimidine bases back to their nucleosides.

Biosynthesis of purines: here are three main processes that contribute to purine nucleotide
biosynthesis (1) Synthesis from amphibolic intermediates (2) Phosphoribosylation of purines
(3) phosphorylation of purine nucleotides.
A number of reactions take place with phosphoribosyl pyrophosphate as initial substrate that
eventually lead to the formation of inosine 5-phosphate that serves as branch point from
which adenine or guanine nucleotides can be formed. In the case of adenine, inosine-5-
phosphate forms adenylosuccinate by the action of adenylosuccinate synthase that requires
Mg
2+
, addition of aspartate and elimination of water. Adenylosuccinate then forms adenosine-
5-phosphate (AMP) by elimination of succinate and participation of adenylosuccinase.
In the case of guanine, inosine-5-phosphate forms xanthosine-5-phosphate by IMP
dehydrogenase and reduction of NAD
+
. Xanthosine monophosphate forms guanosine
monophosphate (guanosine-5-phosphate) by hydrolysis of ATP, donation of nitrogen from
glutamine that forms glutamate and the enzyme transamidinase. Inhibitors of purine
synthesis act as antineoplastic agents.

67

Purine synthesis is mainly regulated at the enzyme PRPP glutamyl amidotransferase (PRPP
5-phosphoribosylamine) which is inhibited by AMP, GMP, and IMP and activated by
PRPP.
61. Catabolism of purine and pyrimidine nucleotides and elimination of the
end-products:

Catabolism of purine and pyrimidine nucleotides:

Pyrimidine: Degradation of pyrimidines involves dephosphorylation and cleavage of
nucleosides. Free bases of pyrimidine result in NH
3
, CO
2
, -alanine. Soluble compounds are
excreted by urine. See paper for reactions.

Purine: See paper for reactions. Purines are catabolised into uric acid that in high
concentrations can lead to gout. About 400-600 mg /day are excreted. Causes of gout are
overproduction of uric acid, under excretion of uric acid in kidneys that might be deposited in
the joints and inflammatory gout might arise as an inflammatory response. Another reason for
gout is the Lech-Nyhan syndrome where there is a lack of hypoxanthine-guanine
phosphoribosyl transferase, an enzyme of the purine salvage. This is an inherited disorder.

68. Membrane structure, the assembly and recycling of membranes.
Specialized structures of plasma membrane lipid rafts, caveols, tight
junctions:

Membrane structure:

Biological membranes consist of a lipid bilayer with a hydrophobic interior and a hydrophilic
exterior. The interior of the cell communicates with the exterior environment by selective
permeabilities of the plasma membrane provided by special transporters and ion channels.
The plasma membrane also exchanges material with the external environment by endocytosis
and exocytosis.
The lipids making up the membrane bilayer are primarily phospholipids (phosphoglycerides,
ceramides), glycosphingolipids (gangliosides, cerebrosides) and cholesterol. Cholesterol
provides rigidity to the cell membrane and is referred to as LDL-cholesterol. Within the
membrane integral and peripheral proteins are present.
Lipid rafts are specialized areas of the exoplasmatic leaflet of cell membrane that is enriched
in cholesterol, sphingolipids, and certain proteins. These lipid rafts are involved in e.g. signal
transduction.
Caveoles may derive from lipid rafts and contain in the majority of cases the protein
caveolin-1. Caveoles may be seen as invaginations of the cell membrane facing the cytosol.
In caveoles proteins can be found that are involved in e.g. insulin receptors, folate receptors
and various other proteins involved in the signal transduction system.
Tight junctions are other structures found on the surface of membranes that consist of
proteins that prevent diffusion of macromolecules between two adjacent cells.

69. Transport across membranes (various types of passive and active
transport mechanisms, characters of transporters and ionophores,

68

examples):

Transport across membranes:

Molecules can passively diffuse through a membrane by simple diffusion or facilitated
diffusion down a concentration gradient. There will be an equalizing of concentrations on
both sides of the membrane. Simple diffusion is the passive flow of a solute from a higher to a
lower concentration. Facilitated diffusion is passive transport of a solute from a higher
concentration to a lower concentration mediated by transporter proteins. These transporter
proteins can involve either certain transporters or ion channels that exist in the biological
membranes that can be classified depending on direction of movement or if it is upstream or
downstream the concentration gradient.
Uniport: Moves one type of molecule bidirectionally.
Cotransport: The transfer of one solute depends on the simultaneous or sequential transfer of
another solute. Includes the groups of symport and antiport.
Symport: Two solutes are moved in the same direction. Example is sodium coupled glucose
transporters.
Antiport: Two molecules are moved in opposite directions. Ex. Na
+
in and Ca
2+
out.
Hydrophilic molecules that cannot pass the cell membrane freely do this by facilitated
diffusion or active transport. Active transport always takes place against an electrical or
chemical gradient requiring energy, in most cases ATP. Both these transport systems use
specific carrier proteins that might be very specific for a certain compound or ion. The rate
at which solutes enter a cell by facilitated diffusion is determined by (1) the concentration
gradient across the membrane (2) the amount of carrier available (3) the affinity of the solute-
carrier interaction and (4) the rapidity of the conformational change for both the loaded and
unloaded carrier.
Transport proteins exist as integral membrane proteins.

Ionophores are molecules that act as membrane shuttles for various ions. Ionophores contain
hydrophilic centres that bind specific ions and are surrounded by peripheral hydrophobic
regions. Example: valinomycin transports K
+


70. Thiamine - the physiological role of TDP (examples of reactions
demanding TDP):

Thiamine:

Thiamine, or vitamin B
1
, is a sulphur-containing water-soluble compound. Sources of
thiamine can be found in yeast, pork and in cereals. Its phosphate derivatives are especially
important in physiological reactions, here are some following examples:

1. In oxidative decarboxylation of pyruvate into acetyl-CoA

2. In alcohol fermentation as a part of the pyruvate decarboxylase complex.


69

3. In the oxidative decarboxylation of -ketoglutarate into succinyl-CoA.

4. In reactions with transketolase in the pentose phosphate pathway.

71. Coenzymes of acyl transferases, transfer of acyls (coenzyme A and
phosphopantetheine, lipoamide, carnitine):

Coenzymes of acyl transferases:

Coenzyme A is transferred in different forms of acyl groups, acetyl-CoA, succinyl-CoA, acyl-
CoA propionyl-CoA etc. Coenzyme A is formed from three components; cysteamine,
pantothenate and adenosine triphosphate. Since it chemically is a thiol it can react with
carboxylic acids to form thioesters and thus act as an acyl group carrier. Acyl-activated
compounds can be seen above.
Phosphopantetheine is an essential prosthetic group of acyl carrier protein that is derived from
coenzyme A.
Lipoamide is a part of the cofactors necessary for oxidative decarboxylation of pyruvate into
acetyl-CoA. First, thiamine diphosphate forms hydroxyethyl thiamine diphosphate by transfer
of carboxyl group. Lipoamide then forms a thioester bond with the remaining acetaldehyde
(after decarboxylation of pyruvate) and then CoA replaces lipoamide and acetyl-CoA is
formed.
Carnitine combines with acyl-CoA (activated fatty acids) to form acylcarnitine that is
permeable to the inner mitochondrial membrane.

72. Methylation and carboxylation - reaction sequences, enzymes and
coenzymes, the roles in metabolism:

Methylation:

Methylation reactions denotes reactions where there is an addition of a methyl group to a
substrate or the substitution of an atom by a methyl group. Methylation performed by
coenzymes includes e.g. tetrahydrofolate that transfer one-carbon fragment, e.g. methyl
groups which bind to either the N5 or N10 atoms. This occurs e.g. in biosynthesis of purine
bases and methylation of uracil to thymine.

Carboxylation:

In carboxylation reactions carboxylic groups are introduced into a substrate, e.g. in the form
of carbon dioxide. Biotin is a cofactor (vitamin B
7
) that is needed in carboxylation reactions
where it alternates between biotin and carboxybiotin, a conversion requiring ATP. Biotin is a
cofactor of the enzyme pyruvate carboxylase that catalyzes the conversion of pyruvate into
oxaloacetate, which is both an anaplerotic reaction of the citric acid cycle and a reaction of the
gluconeogenesis. Another reaction requiring biotin is the conversion of propionyl-CoA into
methylmalonyl-CoA by carboxylation and then hydrolysis of ATP. Methylmalonyl-CoA is
then converted into succinyl-CoA.


70

73. Folate and tetrahydrofolate (structures, relations to 4-aminobenzoate
and action of sulfonamides); one-carbon units - sources, transfer and
interconversions, the utilization):

Folate and tetrahydrofolate:

Folate is a vitamin B
9
that is nutritionally essential since the body cannot synthesize it. It is
composed of pteridine, p-aminobezoic acid (4-aminobenzoic acid, PABA) which is linked via
an amide bond to glutamic acid. A number of drugs interfere with the biosynthesis of folic
acid and tetrahydrofolate. Sulfonamides act as competitive inhibitors of 4-aminobenzoate that
inhibits the formation of folic acid.
Folic acid transfer one-carbon units such as methyl, methylene, methenyl, formyl etc.
Sources of folate in the diet is e.g. from leafy vegetables (spinach), legumes, (peas, beans,
lentils), yeast, and sunflower seeds.
Folic acid is important in DNA and RNA synthesis, methylation in synthesis of purine bases
and thymine, in the synthesis of red blood cells etc. Reactions with tetrahydrofolate and
glycine, serine and choline forms methylene-tetrahydrofolate that is the major point of entry
for one-carbon units. When acting as a methyl donor, methionine is converted into
homocysteine. When homocysteine is regenerated it is performed by methyl-tetrahydrofolate
catalyzed by methionine synthase, a vitamin B
12
dependent enzyme. A deficiency of vitamin
B
12
leads to an accumulation of methyl-tetrahydrofolate, the folate trap. Therefore, a
deficiency of vitamin B
12
leads to a secondary deficiency of folate.

74. L-Ascorbate - sources, utilization in biochemical redox reactions
(examples):

L-Ascorbate:

Ascorbic acid is a hydrophilic sugar acid with antioxidant properties. One form of ascorbic
acid is known as vitamin C. It is especially rich in fruits (citrus fruits) and vegetables
(potatoes etc.). Humans cannot synthesize ascorbic acid from the uronic acid pathway from
glucose metabolism, therefore it is nutritionally essential.
Ascorbic acid has a specific role in Cu
2+
containing hydroxylases, e.g. in dopamine -
hydroxylase, a copper-containing enzyme involved in the synthesis of catecholamines from
tyrosine. During hydroxylation the Cu
+
is oxidized into Cu
2+
, reduction back to Cu
2+
requires
ascorbate.
Ascorbate is also important in regeneration of antioxidants, e.g. tocopherol after it has been
oxidized and needs to be reduced again to gain function again.