Metabolism is the set of chemical rections that occur in a cell, which enable it to keep living,

growing and dividing. Metabolic processes are usually classified as:

 catabolism - obtaining energy and reducing power from nutrients.

 anabolism - production of new cell components, usually through processes that require
energy and reducing power obtained from nutrient catabolism.

There is a very large number of metabolic pathways. In humans, the most important metabolic
pathways are:

 glycolysis - glucose oxidation in order to obtain ATP

 citric acid cycle (Krebs' cycle) - acetyl-CoA oxidation in order to obtain GTP and valuable
intermediates.

 oxidative phosphorylation - disposal of the electrons released by glycolysis and citric acid
cycle. Much of the energy released in this process can be stored as ATP.

 pentose phosphate pathway - synthesis of pentoses and release of the reducing power
needed for anabolic reactions.

 urea cycle - disposal of NH4+ in less toxic forms

 fatty acid -oxidation - fatty acids breakdown into acetyl-CoA, to be used by the Krebs'
cycle.

 gluconeogenesis - glucose synthesis from smaller percursors, to be used by the brain.

Click on the picture to get information on each pathway

Metabolic pathways interact in a complex way in order to allow an adequate regulation. This
interaction includes the enzymatic control of each pathway, each organ's metabolic profile
and hormone control.

Enzymatic control of metabolic pathways

Regulation of glycolysis

Metabolic flow through glycolysis can be regulated at three key points:

 hexokinase: is inhibited by glucose-6-P (product inhibition)

 phosphofructokinase: is inhibited by ATP and citrate (which signals the abundance
of citric acid cycle intermediates). It is also inhibited by H+, which becomes important
under anaerobiosis (lactic fermentation produces lactic acid, resulting on a lowering of
the pH ). Probably this mechanism prevents the cell from using all its ATP stock in the
phosphofrutokinase reaction, which would prevent glucose activation by hexokinase.
It is stimulated by its substrate (fructose-6-phosphate), AMP and ADP (which signal
the lack of available energy), etc.
 pyruvate kinase: inhibited by ATP and acetyl-CoA

Regulation of gluconeogenesis

Flow is regulated in the gluconeogenesis-specific reactions. Pyruvate carboxilase is activated

by acetyl-CoA, which signals the abundance of citric acid cycle intermediates, i.e., a
decreased need of glucose.
Regulation of the citric acid cycle

The citric acid cycle is regulated mostly by substrate availability, product inhibition and by
some cycle intermediates.

 pyruvate dehydrogenase: is inhibited by its products, acetyl-CoA and NADH

 citrate synthase: is inhibited by its product, citrate. It is also inhibited by NADH and
succinyl-CoA (which signal the abundance of citric acid cycle intermediates).
 isocitrate dehydrogenase and -ketoglutarate dehydrogenase: like citrate synthase,
these are inhibited by NADH and succinyl-CoA. Isocitrate dehydrogenase is also
inhibited by ATP and stimulated by ADP. All aforementioned dehydrogenases are
stimulated by Ca2+. This makes sense in the muscle, since Ca2+ release from the
sarcoplasmic reticulum triggers muscle contraction, which requires a lot of energy.
This way, the same "second messenger" activates an energy-demanding task and the
means to produce that energy.

Regulation of the urea cycle

Carbamoyl-phosphate sinthetase is stimulated by N-acetylglutamine, which signals the

presence of high amounts of nitrogen in the body.

Regulation of glycogen metabolism

Liver contains a hexokinase (hexokinase D or glucokinase)with low affinity for glucose which
(unlike "regular" hexokinase) is not subject to product inhibition. Therefore, glucose is only
phosphrylated in the liver when it is present in very high concentrations (i.e. after a meal). In
this way, the liver will not compete with other tissues for glucose when this sugar is scarce,
but will accumulate high levels of glucose for glycogen synthesis right after a meal.

Regulation of fatty acids metabolism

Acyl-CoA movement into the mitochondrion is a crucial factor in regulation. Malonyl-CoA

(which is present in the cytoplasm in high amounts when metabolic fuels are abundant)
inhibits carnitine acyltransferase, thereby preventing acyl-CoA from entering the
mitochondrion. Furthermore, 3-hydroxyacyl-CoA dehydrogenase is inhibited by NADH and
thiolase is inhibited by acetyl-CoA, so that fatty acids wil not be oxidized when there are
plenty of energy-yielding substrates in the cell.

Regulation of the pentose phosphate pathway

Metabolic flow through the pentose phosphate pathway is controled by the activity of
glucose-6-phosphate dehydrogenase, which is controlled by NADP+ availability.

Metabolic profiles of key tissues

Brain
Usually neurons use only glucose as energy source. Since the brain stores only a very small
amount of glycogen, it needs a steady supply of glucose. During long fasts, it becomes able to
oxidize ketone bodies.

Liver

The maintenance of a fairly steady concentration of glucose in the blood is one of the liver's
main functions. This is accomplished through gluconeogenesis and glycogen synthesis and
degradation. It synthesizes ketone bodies when acetyl-CoA is plenty. It is also the site of urea
synthesis.

Adipose tissue

It synthesizes fatty acids and stores them as triacylglycerols. Glucagon activates a hormone-
sensitive lipase, which hydrolizes triacylglycerols yielding glycerol and fatty acids. These are
then released into the bloodstream in lipoproteins.

Muscle

Muscles use glucose, fatty acids, ketone bodies and aminoacids as energy source. It also
contains a reserve of creatine-phosphate, a compound with a high phosphate-transfer potential
that is able to phosphorilate ADP to ATP, thereby producing energy without using glucose.
The amount of creatine in the muscle is enough to sustain about 3-4 s of exertion. After this
period, the muscle uses glycolysis, first anaerobically (since it is much faster than the citric
acid cycle), and later (when the increased acidity slows phosphofrutokinase enough for the
citric acid cycle to become non-rate-limiting) in aerobic conditions.

Kidney

It can perform gluconeogenesis and release glucose into the bloodstream. It is also responsible
for the excretion of urea, electrolytes, etc. Metabolic acidosis may be increased by the action
of the urea cycle, since urea synthesis (which takes place in the liver) uses HCO3-, thereby
further lowering blood pH. Under these circunstances, nitrogen may be eliminated by the joint
action of kidney and liver: excess nitrogen is first incorporated in glutamine by glutamine
synthetase. Kidney glutaminase then cleaves glutamine in glutamate e NH3, which the kidney
immediately excretes. This process allows nitrogen excretion without affecting blood
bicarbonate levels.

Hormone control
Hormone control is mainly effected through the action of two hormones synthesized by the
pancreas: insulin and glucagon. Insulin is released by the pancreas when blood glucose levels
are high, i.e., after a meal. Insulin stimulates glucose uptake by the muscle, glycogen
synthesis, and triacylglyceride synthesis by the adipose tissue. It inhibits gluconeogenesis and
glycogen degradation. Glucagon is released by pancreas when blood glucose levels drop too
much. Its effects are opposite those of insulin: in liver, glucagon stimulates glycogen
degradation and the absorption of gluconeogenic aminoacids. It inhibits glycogen synthesis
and promotes the release of fatty acids by adipose tissue.
Blood glucose levels are kept at approximately constant levels around 4-5 mM. Glucose
enters cells by facilitated diffusion. Since this process does not allow the cell to contain
glucose at a higher concentration than the one present in the bloodstream, the cell (through the
enxyme hexokinase) chemically modifies glucose by phosphorylation:

Since the cell membrane is impermeable to glucose-6-phosphate, this process effectively

"traps" glucose inside the cell, allowing the recovery of more glucose from the bloodstream.
Glucose-6-phosphate will be used in glycogen synthesis (a storage form of glucose) ,
production of other carbon compounds by the pentose-phosphate pathway, or degraded in
order to produce energy- glycolysis.

In order to be used for energy production, glucose-6-phosphate must first be isomerized in

fructose-6-phosphate. Fructose-6-phosphate is again phosphorylated to fructose-1,6-
bisphosphate, in a reaction catalyzed by phosphofructokinase. This is the commited step of
this metabolic pathway: from the moment glucose is transformed into fructose-1,6-
bisphosphate it must proceed through glycolysis.
Cells contain 2 phosphofructokinase forms: PFK 1 (which produces fructose-1,6-
bisphosphate) and PFK 2. PFK 2 produces fructose-2,6-bisphosphate (F-2,6-BP), which is an
activator of PFK 1 and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase.
F-2,6-BP therefore prevents gluconeogenesis from occuring at the same time as glycolysis.
When blood glucose levels are low, pancreas releases glucagon. Glucagon activates the
hydrolysis of fructose-2,6-bisphosphate, which relieves the inhibition of gluconeogenesis, and
depresses glycolysis.

After this conversion, an inverse aldolic addition cleaves fructose-1,6-bisphosphate in two

three-carbon molecules :

Both molecules (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate) can easily be

interconverted by isomerization. A single metabolic pathway is therefore enough to degrade
both. This is why glucose-6-P was first isomerized to fructose-6-P: glucose-6-P breakdown
through an inverse aldol addition would yield two quite different molecules (of two and four
carbons, respectively), which would have to be degraded through two different pathways.

Aldehydes have very low redox potentials(around -600 to -500 mV). Oxidation of
glyceraldehyde-3-phosphate by NAD+ (E0=-320 mV) is therefore quite spontanteous. Indeed,
it is so exergonic that it can be used to produce ATP (ATP production from ADP and Pi can
be performed if coupled to a two-electron redox reaction with a potential difference of at least
160 mV). ATP production happens through two consecutive steps: in the first step,
gliceraldehyde-3-phosphate oxidation to a carboxylic acid is coupled to the phosphorylation
of the produced carboxylic acid.
Phosphorilated acids (as well as phosphoenols and phosphoguanidines) contain very energetic
phosphate groups: hydrolysis of these phosphate groups yields with very significant resonance
stabilization. Therefore, the phosphate group attached to carbon 1 in 1,3-bisphosphoglycerate
can be easily transferred to ADP, in order to produce ATP.

3-Phosphoglycerate is isomerized to 2-phosphoglycerate, which after dehydration (i.e. losing

H2O) yields a phosphoenol:

Due to its high phosphate transfer potential phosphoenolpyruvate can transfer a phosphate
group to ADP:

Two ATP molecules are used in glycolysis, and four ATP are produced. NAD+ must be
continuously regenerated, otherwise glycolysis will stop, since NAD+ is a substrate in one of
the reactions. Under aerobic conditions, NADH transfers its two electrons to the electron-
transport chain . In animal cells, in the absence of O2 NADH transfers its electrons to the end-
product of glycolysis (pyruvate), yielding lactate. This is called fermentation : an internally
balanced degradation, i.e., a process that uses one of its products as the final acceptor of the
electrons it releases.

The human body has two main ways to keep constant blood glucose levels between meals:
glycogen degradation and gluconeogenesis. Gluconeogenesis is the synthesis of glucose from
other organic compounds (pyruvate, succinate, lactate, oxaloacetate, etc. Most of the reactions
involved are quite similar to the reverse of glycolysis. Indeed, almost all reactions in glycolyis
are readily reversible under physiological conditions. The three exceptions are the reactions
catalyzed by :

 pyruvate kinase

 phosphofrutokinase

 hexokinase
In gluconeogenesis, every one of these steps is replaced by thermodinamically favorable
reactions. Among these three reactions, phosphoenolpyruvate synthesis from pyruvate is the
most energy-demanding, since its G is rather positive. In order to overcome this
thermodynamic barrier, the reaction will be coupled to a decarboxylation, a strategy often
used by the cell to displace an equilibrium towards the formation of products, as it will also be
observed in several reactions in the citric acid cycle. Since both pyruvate and
phosphoenolpyruvate(PEP) are three-carbon compounds, pyruvate must be carboxylated to a
four-carbon compound, oxaloacetate (OAA), before such a decarboxylation can happen. The
enzyme responsible for pyruvate carboxylation (pyruvate carboxylase) is present inside the
mithocondrial matrix, and contains biotin, a CO2-activating cofactor. The energy required for
the carboxylation comes from from the hydrolysis of ATP. Oxaloacetate decarboxylation
releases the energy needed to enable C2 phosphorylation by GTP, yielding
phosphoenolpyruvate (in a reaction catalyzed bynuma phosphoenolpyruvate carboxykinase
- PEPCK).

Oxaloacetate produced by the pruvate carboxylase cannot cross the mithochodrial membrane.
It can only leave the mithochondrion after conversion to malate or aspartate. The choice of the
process depens on the availability of cytoplasmic NADH (needed for gluconeogenesis). If
there is enough NADH in th cytoplasm (e.g. when lactate is being used as gluconeogenic
substrate) oxaloacetate will be transaminated to aspartate. Otherwise, OAA will be reduced to
malate in the mithochondrial matrix. The mithochondrial membrane is permeable to malate,
which moves into the cytoplasm, where it can be oxidized to oxaloacetate with concommitant
production of NADH. Oxaloacetate can then be decarboxylated to PEP by the cytoplasmic
PEPCK. Some tissues also contain a mithochondrial PEPCK.

In gluconeogenesis, the reactions catalyzed by phosphofructokinase and hexokinase are

replaced by hydrolytic reactions. Instead of phosphorylating ADP to ATP (the exact reverse
of glycolysis, yet thermodynamically not favorable under physiological conditions),
phosphate is released by hydrolysis:

Fructose-1,6-bisphosphatase is present in almost all tissues, but glucose-6-phosphatase is only

present in liver and kidney, which allows these organs to supply glucose to other tissues:

During intese physical exercise, lactate produced in the muscles is sent to the bloodstream,
and can be used by the liver as a gluconeogenic substrate. Although 6 ATP are used by the
liver for each new glucose synthesized and only 2 ATP per glucose are released in the muscle
under anaerobic conditions, this "lactate cycle" is advantageous to the organism, since it
allows the maintenance of the anaerobic exercise for a little longer (and this can be crucial for
survival, e.g., by allowing a prey to outrun its predator, or a predator to keep chasing its prey).
Pyruvate produced by glycolysis still contains a lot of reducing power (check each of its
carbon atoms' oxidation state and compare it with carbon's oxidation state in CO2). This
reducing power will be harnessed by the cell through the citric acid cycle. First, pyruvate is
decarboxylated to acetyl-CoA, an activated form of acetate (CH3COO-)

This reaction is catalyzed by pyruvate dehydrogenase, a very complex enzyme with several
cofactors: lipoamide, FAD, coenzyme A. Thioester bond (S-C=O) hydrolysisis very
exergonic, and therfore its formation demands energy. That energy comes from pyruvate
decarboxylation (pyruvate contains three carbon atoms, and the acetyl portion of acetyl-CoA
only contains two: the carboxylate group left as CO2). Energy from decarboxylations is often
used bt the cell to push an equilibrium towards product formation, as can be seen in several
reactions in the citric acid cycle and gluconeogenesis.

In the first reaction of the citric acid cycle, acetyl-CoA attacks oxaloacetate, yielding citrate,
in an aldol addition. Thioester hydrolysis helps to displace equilibrium towards product
formation:
Citrate is then isomerized to isocitrate, which is then decarboxylated to -ketoglutarate. If
citrate had not been isomerized to isocitrate, this decarboxylation would yield a branched
carbon compound, much harder to metabolize.

a-ketoglutarate is a -ketoacid, i.e., it contains a carbonyl group adjacent to a carboxylic acid.

We can predict that it will react like pyruvate, i.e., that its decarboxylation may yield enough
energy to enable the formation of a thioester bond with coenzyme A. And this indeed occurs...
The enzyme involved (-ketoglutarate dehydrogenase), is quite similar to pyruvate
dehydrogenase in composition, cofactors and mechanism.

Like every thioester bond, the one present in succinyl-CoA is quite energetic. Its hydrolysis
will be the only step in the citric acid cycle where direct production of ATP (or equivalent)
occurs.
Like oxaloacetate, succinate is a four-carbon product. The last reactions of the citric acid
cycle will regenerat oxaloacetate from succinate. Succinate is first oxidized to fumarate, by
the succinate dehydrogenase complex (also known as complex II), which is present in the
amtrix side of the inner mitochondrial membrane. The redox potential of the oxidation of a C-
C single bond to a C=C double bond (alkanes to alkenes) is too high to enable the involved
elevtrons to be accepted by NAD+ (E0=-320 mV). the cell will terefore use FAD (E0= 0 mV)
as electron acceptor. Fumarate hydration yields malate, which can be oxidized to
oxaloacetate, thus closing the cycle.A similar sequence of reactions happens in fatty acids -
oxidation.

The end result of the citric acid cycle is therefore:

Acetyl-CoA + oxaloacetate + 3 NAD+ + GDP + Pi +FAD --> oxaloacetate + 2 CO2 + FADH2

+ 3 NADH + 3 H+ + GTP

Besides being the most important building blocks of proteins aminoacids can also be used as
precursors of nitrogen-containing molecules: hemes, nucleotides, glutathione,
physiologically-active amines, etc.

Excess diet aminoacids are neither stored nor excreted as such: they are converted in
pyruvate, oxaloacetate, -ketoglutarate, etc. Therefore, aminoacids are also precursors of
glucose, fatty acids, and ketone bodies, and can be used for energy production.

Aminoacid conversion involves the removal of the amine group (eamination), the
incorporation of the ammonia produced in this reaction into urea for excretion and the
conversion of the carbon skeletons into metabolic intermediates.

Deamination of most aminoacids involves a transamination step, i.e. the transfer of their
amino groups to a -ketoacid, thereby producing the aminoacid equivalent of the original -
ketoacid and the -ketoacid equivalent of the original aminoacid. The amine acceptor is
usually -ketoglutarate, which is converted to glutamate:

Aminotransferases use pyridoxal-5'-phosphate, a derivative of vitamin B6. Pyridoxal is also

involved in reactions of aminoacid decarboxilation and removal of their sidechains. It is also
the cofactor used by glycogen phosphorylase, although in this case the reaction mechanism is
different. Aminotransferases are specific for each type of aminoacid, and produce the
corresponding -ketoacids. However, most accept only -ketoglutarate or (to a minor extent)
oxaloacetate as amine acceptor, yielding glutamate or aspartate, respectively. Therefore, most
amine groups will eventually end in glutamate or aspartate, which can be interconverted by
glutamate-aspartate aminotransferase.

A set of muscle aminotransferases use pyruvate (which is also a -ketoacid) as amine

acceptor, producing the corresponding aminoacid, alanine . Upon release to the bloodstream,
alanine is taken up by the liver, which transaminates it back to pyruvate, to be used in
gluconeogenesis. The glucose produced in this process will be oxidized to pyruvate (and
eventually lactate or CO2, depending on the conditions) by the muscle, thereby completing the
alanine cycle. The released amino group will be used in urea synthesis. The net result of the
alanine cycle is the transport of nitrogen from muscle to liver.

Transamination does not yield nitrogen release from aminoacids: it simply transfers the amino
groups from a large variety of aminoacids into two or three aminoacids (glutamate, aspartate
and alanine). Deamination is performed for the most part by glutamate dehydrogenase, a
mitochodrial enzyme unique for its ability to use either NAD+ or NADP+.

Nitrogen released by this reaction as ammonia must be excreted. Many water-living animals
excrete it without modification. Other animals with less plentiful water supplies convert
ammonia into less toxic products that need less water to be excreted. One of these products is
urea.

The reasons for ammonia toxicity are not yet fully understood, but it is known that in high
concentrations, ammonia reacts with glutamate yielding glutamine, in a reaction catalyzed by
glutamine synthetase.

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In order to replenish glutamate stocks, other aminoacids react with -ketoglutarate by
transamination. As a result of both reactions, -ketoglutarate e glutamate progressively
become exhausted, with very harmful consequences for neuron function (since glutamate is a
precursor of neurotransmitters, and citric acid cycle operation is dependent on a steady level
of intermediates like -ketoglutarate).

Urea is synthesized in the liver, which secretes it to the bloodstream, whence it will excreted
by the kidney. The global reaction of the urea cycle is:

The first step is the synthesis of carbamoyl-phosphate, an activated form of nitrogen:

Carbamoyl is afterwards tranferred to ornithine, yielding citrulline. Bithe these molecules are
"special" aminoacids, i.e., aminoacids which are not in the restricted set of 21 aminoacids
used in protein synthesis.

After these two reactions (which occur inside the mitochondrion), citrulline is transferred to
the cytoplasm, where the remainder of the cycle happens.

The second nitrogen atom in urea comes from aspartate:

In this reaction, ATP is hydrolized to AMP, instead of ADP (as usually happens). Since AMP
can accept a phosphate group from ATP, yielding 2 ADP, hydrolisis of ATP to AMP is
equivalent to the hydrolysis of 2 ATP to 2 ADP.

Argininosuccinate can be cleaved into arginine and fumarate:

Upon entry into the mitochondrion, fumarate can react in the citric acid cycle to produce
NADH and oxaloacetate, which can be converted into aspartate by transamination.

Arginine hydrolysis yields urea and ornithine, which can restart the cycle after entering the
mitochondrion.
The urea cycle has a high energy cost, equivalent to the hydrolysis of 4 ATP to 4 ADP.
However, this cost can be regained in the electron-transport chain, since the NADH produced
in glutamate deamination and in the oxidation of fumarate to oxaloacetate are equivalent to
about 6 ATP.

Blood glucose levels are kept at approximately constant levels around 4-5 mM. Glucose
enters cells by facilitated diffusion. Since this process does not allow the cell to contain
glucose at a higher concentration than the one present in the bloodstream, the cell (through the
enxyme hexokinase) chemically modifies glucose by phosphorylation:

Since the cell membrane is impermeable to glucose-6-phosphate, this process effectively

"traps" glucose inside the cell, allowing the recovery of more glucose from the bloodstream.
Glucose-6-phosphate will be used in glycogen synthesis (a storage form of glucose) ,
production of other carbon compounds by the pentose-phosphate pathway, or degraded in
order to produce energy- glycolysis.

Large ammounts of glucose-6-P inside the cell cause and increase of the osmotic pressure. In
these conditions, water will tend to flow into the cell, increasing its colume and (eventually)
lysing it. In order to prevent this, the cell stores glucose-6-P as a polymer: glycogen.
Glycogen is a sparsely soluble (and therefore osmotically inactive) branched polyssacharide,
composed of glucose monomers joined through glycosidic bonds of the type -1,4 and -1,6
(in branching points) :
In order to be used for glycogen synthesis, glucose-6-fosfato is first isomerized to glucose-1-
fosfato by the enzyme fosfoglucomutase.

Addition of glucose-1-P to the 4' carbon of a glycogen chain is not favored

thermodinamically, since the phosphate transfer potential of C-O-P bonds is quite low.
Glucose-1-P will therefore be activated, i.e., transformed into a species with high phosphate
transfer potential. This is accomplished by reaction with uridine triphosphate(UTP, an analog
of ATP, with uridine replacing adenine).
By itself, this reaction seems not to be thermodynamically favourable. However,
pyrophosphate (PPi) released in this reaction can be hydrolyzed by the ubiquitous enzyme
pyrophosphatase, in a very exergonic reaction. Removal of PPi pushes the equilibrium
towards the formation of UDP-glucose, which illustrates the general principle that a very
exergonic reaction can be coupled to an otherwise unfavourable reaction in order to make it
spontaneous.

UDP-glucose has a high phosphate transfer potential, and this allows it to donate glucose to
the 4' end of a glycogen chain, in a reaction catalyzed by glycogen synthase:

Glycogen synthase can only add glucose to pre-existent glycogen chains,i.e, it is unable to
start the synthesis of a new glycogen molecule. Glycogen synthesis is started by the addition
oa a glucose molecule to a tyrosine residue present in the active site of a protein called
glycogenin. After addition of around seven more glucose molecules, the new glycogen chain
is ready to be acted upon by glycogen synthase

Branching points are created by a "branching enzyme". This enzyme acts upon linear
stretches of glycogen with at least 11 glucose molecules. Branching enzyme (amylo(1,4 --
>1,6)-transglycosylase) transfers 7 glucose molecules-long terminal segments of glycogen to
the OH group of carbon 6 of a glucose reidue (in the same or in another chain). Branching
points must be at least 4 glucose molecules apart from each other.

Glycogen degradation
Glycogen is degraded by the sequential action of three enzymes:

 glycogen phosphorylase cleaves (1-4) bonds with inorganig phosphate(Pi). It can

only cleave glucose residues 4 (or more) glucose residues away from a branching
point . It uses pyridoxal, a vitamin B6 derivative, as cofactor.

A glycogen molecule with branches of only four glucose molecules ("limit-dextrin") cannot
be further degraded by glycogen phosphorylase alone. It needs another enzyme:

 glycogen debranching enzyme: transfers three glucose residues from a limit branch
to another. The last residue in the branch (with a (1-6) glycosidic bond) is removed
by hydrolysis, yielding free glucose and debranched glycogen. Hydrolysis of this
residue is catalyzed by the same debranching enzyme.
 Glycogen phosphorylase is much faster than the debranching enzyme, and therefore
the outer branches of glycogen are degraded bery rapidly in muscle when much energy
is needed. Glycogen degradation beyond this point demands the action of the
debranching enzyme and is therefore slower, which partly explains the fact that the
muscle can only perform its maximum exertion during a few.

 phosphoglucomutase: catalyzes the isomerization of glucose-1-P to glucose-6-P, and

vice-versa:

Glucose 6-phosphate can then be used in glycolysis. Unlike muscle, liver (and to a smaller
degree, kidney) contains glucose-6-phosphatase, a hydrolytic enzyme catalyzing glucose-6-
phosphate dephosphorylaton that allows it to supply glucose to other tissues:
Fatty acids -oxidation
Most energy reserves in the body are stored as triacylglycerides, which can be hydrolyzed to
glycerol and fatty acids through the action of lipases:

Glycerol can be metabolized by glycolysis upon oxidation (in the outer face of the inner
mitochodrial membrane) to dihydroxyacetone phosphate. Both electrons are taken up by
ubiquinone (Q), and are fed into the electron transport chain.

Fatty acids follow a different pathway: -oxidation, which takes place in the mitochondrion.
Before entering the mitochondrion, fatty acids must be activated. The activation reaction
happens in the cytoplasm, and it consists on the transformation of the fatty acid into its acyl-
Coa derivative. As we have seen in the citric acid cycle, thioester bonds are very energetic.
Therefore, an ATP gets hydrolyzed (to AMP, which is equivalent to the hydrolysis of 2 ATP
to 2 ADP) in the process.

The mithochondrial inner membrane is impermeable to acyl-CoAs. In order to get inside,

these will react with a "special" aminoacid, carnitine, releasing CoA. Sterified carnitine is
transported into the mitochondial matrix by a specific membrane-bound transport complex.
Inside the mitochondrion, carnitine transfers the acyl group to another CoA molecule. Free
carnitine returns to the cytoplasm through the same transporter complx. In this process, no net
CoA transport into the mitochondrion occurs: separate cytoplasmic and mitochondrial CoA
pools are kept .

Fatty acids -oxidation is a cyle composed of three consecutive reactions, which are identical
to the last part of the citric acid cycle: dehydrogenation, hydration of the newly formed C=C
double bond, and oxidation of the alcohol to a ketone:

From the product of these reactions, the enzyme thiolase releases acetyl-CoA and an acyl-
CoA with two carbon atoms less than the original acyl-CoA.

Insaturated fatty acids follow a similar pathway, although new enzymes are needed to deal
with the C=C double bonds. If a double bond lies on an odd C atom, Δ3, Δ2-enoyl-CoA
isomerase is needed: this enzyme transfers the double bond from C3 to C2, thereby allowing
β-oxidation. In this β-oxidation cycle no FADH2 is formed.
When the double bond lies on an even-numbered carbon, 2,4-dienoyl-CoA reductase is
needed, since the presence of cunjugated double bonds makes hydration more favorable on
carbon 4, rather than on the "right" carbon (2). 2,4-dienoyl-CoA reductase uses two electrons
from NADPH to reduce the Δ4, Δ2 system, and form a single double bond on carbon 3.
Oxidation then follws the same procedure used for fatty acids bearing a double bond on an
odd-numbered carbon..

Succesive rouds of the cycle eventually lead to the total degradation of even-chain fatty
acids in acetyl-CoA, which can be completely oxidized to CO2 through the citric acid cycle:
even-chain fatty acids cannot be used for net synthesis of oxaloacetate, and therefore are not a
substrate for gluconeogenesis.

In the last round of -oxidation, odd-chain fatty acids yield acetyl-CoA and propionyl-CoA.
In order for propionyl-CoA to be used by the citric acid cycle it must acquire an extra carbon
atom, and this is accomplished by carboxilation. Methylmalonyl-CoA formed in this reaction
is then rearranged succinyl-CoA, in a cobalamine (a vitamin B12 derivative)-assisted reaction.
Succinyl-CoA is an intermediate in the citric acid cycle and also a precursor of heme
biossynthesis. A vitamin B12 deficiency therefore impairs the ability to synthesize heme and
may eventually lead to the onset of pernicious anemia . This disease is usually caused by the
lack of ability to retrieve cobalamin from the nutrients in the stomach, and is observed in
predisposed individuals in old age. Before modern methos of cobalamin production, treatment
of this disease consisted in the daily uptake of large amounts of raw liver, which is a cery
good reservoir of this heat-labile vitamin. The almost exclusive onset of the disease in old
patients is a consequence of the presence in our own liver of a B12 stock enough for about 3-5
years, so that the effects of an impairment of its absorption will be very delayed.

Succinyl-CoA can be oxidized by the citric acid cycle to malate, which after moving into the
cytoplasm can be used in gluconeogenesis. In the cytoplasm, malate can also be
decarboxylated to pyruvate by the malic enzyme, with cincommitant NADPH production:

Pyruvate formed in this reaction can enter the mitochondrion and be oxidized completely to
CO2 by the citric acid cycle.

Peroxisomal degradation of fatty acids

Peroxisomes are small organelles where the initial steps of -oxidation of very long chain
fatty acids occur. The major differences between mitochondrial and peroxisomal -oxidation
are:

 Fatty acids diffuse freely into the peroxisome: they do not need to be transported by
carnitine. The oxidation product move into the mitochondrion after esterifying
carnitine.
 acyl CoA oxidation used oxygen instead of FAD as electron acceptor, yielding
hydrogen peroxide.
  peroxisomal thiolase is all but inactive with acyl-CoA shorter than 8
carbons.Peroxissomal fatty acid oxidation is therefore incomplete.

Ketogenesis
Much of the acetyl-CoA produced by fatty acid -oxidation in liver mitochodria is converted
in acetoacetate and -hydroxybutyrate (also known as ketone bodies). These molecules can
be used by heart and skeletal muscle to produce energy. Brain, which usually depends on
glucose as sole energy source, can also use ketone bodies during a long fasting period (larger
than two or three days). Ketogenesis (ketone bodies synthesis) begins with the condensation
of two acetyl-CoA molecules to form acetoacetyl-CoA:

Condensation of another acetyl-CoA molecule yields 3-hydroxy-3-methyl-glutaryl-CoA

(HMG-CoA). The basic mechanism of this reaction is identical to the condensation of
oxaloacetate with acetyl-CoA to produce citrate, the first step in the citric acid cycle.

HMG-CoA is afterwards cleaved in acetoacetate and acetyl-CoA:

Acetoacetate moves into the bloodstream and gets distributed to the tissues. Once absorbed, it
reacts (in mitochondria) with succinyl-CoA, yielding succinate and acetoacetyl-CoA, which
can be cleaved by thiolase into two molecules of acetyl-CoA.

Fatty acids synthesis

When acetyl-CoA is abundant, liver and adipose tissue synthesize fatty acids. The syntheis
pathway is quite similar to the reverse of -oxidation, but presents several imporatant
differences:

 it takes place in the cytoplasm, rather than in the mitochondrion.

 uses NADPH as electron donor
 the acyl carrier group is ACP (Acyl Carrier Protein), instead of coenzyme A.

Fatty acids synthesis uses acetyl-CoA as main substrate. However, since the process is quite
endergonic acetyl-CoA must be activated, which happens through carboxylation. Like other
carboxylases (e.g., those of pyruvate or propionyl-CoA), Acetyl-CoA carboxilase uses biotin
as a prosthetic group.

Malonyl-CoA is afterwards transferred to the acyl carrier protein (ACP), yielding malonyl-
ACP, which will condense with acetyl-ACP (sinthesized likewise from acetyl-CoA).
In animals, every step of palmitic acid (the 16-carbon saturated fatty acid) synthesis is
catalyzed by fatty acid synthase, a very large enzyme with multiple enzymatic activities.
Butiryl-ACP produced in the first reaction will be transformed in butyl-ACP (the 4-carbon
acyl-ACP). The reaction sequnce is the reverse of -oxidation, i.e., reduction, dehydration and
hydrogenation:

Butyl-ACP can afterwards condense with another malonyl-ACP molecule. After seven rounds
of this cycle palmitoyl-ACP is produced. Palmitoyl-ACP hydrolysis yields palmitic acid. The
stoichiometry of palmitic acid synthesis is therefore:

Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 7 H+ ---> palmitic acid + 7 CO2 + 14 NADP+

+ 8 CoA + 6 H2O

Longer (or unsaturated) fatty acids are produced from palmitic acid by elongases and
desaturases.

Fatty acid synthesis happens in the cytoplasm, but acetyl-CoA is produced in the
mitochondrion. Therefore acetyl-CoA must cross the inner mitochondrial membrane before it
can be used in fatty acid synthesis. This is performed by the citrate shuttle: citrate is formed
in the mitochondrion by condensing acetyl-CoA with oxaloacetate and diffuses through the
membrane into the cytoplasm, where it gets cleaved by citrate-lyase into acetyl-CoA and
oxaloacetate, whic, upon reduction to malate, can return to the mitochondrial matrix. Malate
can also be used to produce part of the NADPH needed for fatty acid synthesis, through the
action of the malic enzyme. The remainder of the NADPH needed for fatty acid synthesis
must be produced by the pentose phosphate pathway.
In order to perform its anabolism, a cell needs not only energy (ATP): it also needs reducing
power, under the form of NADPH. NADPH can be produced during glucose-6-P oxidation
through a pathway distinct from glycolysis, the pentose-phosphate pathway. This pathway
is very active in tissues involved in cholesterol and fatty acid (liver, adipose tissues, adrenal
cortex, mammal glands). This pathway also produces ribose-5-P, the component sugar of
nucleic acids.

Glucose-6-P's first carbon is first oxidized to a lactone (a cyclic carboxylic acid). Two
electrons are released in this oxidation, and reduce one molecule of NADP+ to NADPH. The
ring is then open by reacting with water:

Gluconate decarboxylation releases two more electrons, which reduce another NADP+
molecule. A five-carbon sugar, ribulose-5-phosphate, is produced in the reaction. By
isomerization, ribulose-5-P is transformed in ribose-5-P. (In the figure, differences between
both isomers are highlighted in green).
What will happen next depends on the needs of the cell: if its needs for NADPH outweigh
those for ribose-5-P, its carbon atoms can be "recycled". This proceeds through three
reactions, which form the non-oxidative part ot the pentose-phosphate pathway. In the first
reaction, ribose-5-P will accept two carbon atoms from xylulose-5-P (obtained by
epimerization of ribulose-5-P), yielding sedoheptulose-7-P and glyceraldehyde-3-P:

Sedoheptulose-7-P transfers three carbons to glyceraldehyde-3-P, yielding fructose-6-P and

erythrose-4-P:

Erythrose-4-P then accepts two carbon atoms from a second molecule of xylulose-5-P,
yielding a second molecul of fructose-6-P and a glyceraldehyde-3-P molecule:
The balance of these three reactions is:

2 xylulose-5-P + ribose-5-P -----> 2 fructose-6-P + glyceraldehyde-3-P

Fructose-6-P and glyceraldehyde-3-P can be degraded by glycolysis in orer to produce

energy, or recycled through gluconeogenesis to regenerate glucose-6-P. In the latter case,
through six consecutive cycles of the pentose-phosphate pathway and gluconeogenesis one
glucose-6-P molecule can be completely oxidized to six CO2 molecules, with concommitant
production of 12 NADPH molecules. When the demand for ribose-5-P is larger than tyhe
demand for NADPH, the non-oxidative part of the pentose-phosphate pathway can operate "in
reverse", yielding three ribose-5-P from two fructose-6-P and one glyceraldehyde-3-P.

Enzymes relay the electrons released by substrate oxidation to special molecules we

call electron acceptors. Electron acceptors may be organic or inorganic, and the most
common examples thereof are NAD+ and FAD. Each of these molecules ca accept two
electrons, yielding NADH+H+ and FADH2, respectively. Since cellular amounts of NAD+ and
FAD are very small, special mechanisms are needed in order to convert NADH+H+ and
FADH2 back into NAD+ and FAD. This is performed through electron transfer from
NADH+H+ and FADH2 to other molecules, which may occur through
either fermentation or respiration. Contrary to general belief, the distinction between these
two processes does not lie on a requirement for O2!

Fermentation

In fermentation, NADH (or FADH2) donates its electrons to a molecule produced by the
same metabolic pathway that produced the electrons carried by NADH (or FADH2). For
instance, during intense physical exercise by muscles, NADH generated through
glycolysis transfers its electrons to pyruvate (an organic molecule produced by
glycolysis), yielding lactate.
(The relationship between the pH drop in muscles during lactate production and the
occurrence of cramps is discussed in detail in these two papers). This process is called lactic
fermentation . Many other kinds of fermentation have been found in microorganisms, and the
most well-known among these is alcoholic fermentation:

Respiration

In respiration, the final acceptor of NADH (or FADH2) electrons is not a product of the
metabolic pathway that released the electrons carried by NADH (or FADH2). Many
microorganisms use SO42-, SeO42- ,NO3-, NO2-, NO, U6+ (uranium), Fe3+, H+, etc. as final
electron acceptors. Mammals use O2, and their respiration is therefore
called aerobic respiration. Aerobic respiration happens in the inner mitochondrial
membrane, which contains the relevant electron-transfering protein complexes. each of
these complexes accepts electrons from a molecule and transfers them to a different
compound, and the full assembly is therefore termed theelectron transport chain:

 NADH dehydrogenase or complex I. In mammals, this complex contains more

than twenty polypeptide chains, many of which with no known function. This
complex accepts two electrons from NADH+H+ and transfer them, through Fe-S
clusters, to a lipophylic molecule, ubiquinone (Q), which gets converted to
ubiquinol (QH2). In this protein complex, electron transfer releases enough
energy to transfer protons (H+) from the mitochondrial matrix to the
intermembrane space, decreasing its pH relative to the matrix.(More details,
including a three-dimensional structure, are available here)
 sucinate dehydrogenase or complex II. This is the sole membrane-bound enzyme in
the citric acid cycle. It oxidizes succinate to fumarate and transfers both released
electrons to FAD, yielding FADH2. As in complex I, these electrons ultimately get
transfered to ubiquinone.(More details, including a three-dimensional structure,
are available here)
 cythocrome bc1 or complex III. It accepts electrons from ubiquinol (generated by
complexes I e II), and transfers them to cythocrome c, a small solule protein
 present in the intermenbrane space. (More details, including a three-dimensional
structure, are available here).
 cythocrome c oxidase or complex IV. It receives four electrons from
cythocrome c and transfers them to O2,thereby reducing it to two water
molecules. (More details, including a three-dimensional structure, are
availablehere)

In complexes I, III and IV , electron

transfer releases enough energy to
transfer H+ from the mitochondrial
matrix to the intermembrane space.
This causes an increase of
H+concentration (and electric potential)
in the intermembrane s+ace, i.e. a
larger chemical potential of H+ in the
intermembrane space relative to the
matrix. However, when we have two
solutions with different concentrations
on both sides of a membrane, solute
tend to diffuse from the regions of
higher chemical potential to areas of
lower potential (for a neutral species,
this is equivalent to moving from areas
of higher concentration to areas of
lower concentration).

The inner mitochondrial membrane is not

permeable to H+. Under normal
conditions, the only way for protons to
flow back to the matrix is through a
special protein: ATP synthetase. This complex protein contains two major portions: an
intermembrane proton channel (F0) coupled to a catalytic protein complex (F1) facing the
mitochondrial matrix. The F1 portion contains several subunits with different functions, and
converts the energy released by the return of protons to the matrix into chemical energy used
to synthesize ATP from ADP and Pi.

NADH is unable to cross the mitochondrial membrane. There are therefore mechanisms to
transfer electrons from NADH molecules produced in the cytoplasm during glycolisis to the
electron transport chain. These are:
 the malate-aspartate shuttle (which also works in gluconeogenesis): NADH transfers
its electrons to oxaloacetate, converting it to malate. Malate can enter the
mitochondrion, where it is dehidrogenated to oxaloacetate and transfer is electrons to
NAD+. This NADH then transfers its electrons to teh electron transport chain through
complex I. Through this shuttle, approxiamtely 3 ATP are produced from each
cytoplasmic NADH.

 the glycerol-3-P shuttle. In this shuttle, which is very active in brown adipose tissue,
cytoplasmic NADH transfer its electrons to the glycolytic intermediate DHAP
(dihydroxyacetone phosphate). DHAP is converted to glycerol-3-P, which donates its
electrons to ubiquinone through a FAD-linked glycerol-3-P dehydrogenase located in the
outer face of the inner mitochondrial membrane. Through this shuttle, approxiamtely 2
ATP are produced from each cytoplasmic NADH.
The amount of ATP produced by ATP synthase is therefore related to the difference in
H+ concentration across the membrane. Since NADH oxidation causes prroton efflux
from the matrix in three protein complexes (I, III e IV), whereas FADH2 oxidation to
FAD is only accompanied by such an efflux in two complexes (III e IV), more ATP can
be produced from NADH than from FADH2. Oxidation of a NADH molecule produces
almost 3 ATP, and FADH2 oxidation yields almost 2 ATP.

Mitochondrial respiration may occur without ATP production, as long as the released protons
are able to return to the matrix without passing through the ATP synthetase. This can happen
e.g. if ionophores (lipid-soluble molecules with the ability to transport ions) are added to the
mitochondria. In brown adipose tissue, a special protein (thermogenin) forms a proton
channel in the mitochondion inner membrane. The flow of protons back into the matrix
through this protein instead of ATP synthetase is responsible for the heat generation
characteristic of this tissue.