Overzicht metabolisme Macromoleculen

8/7/2019 Overzicht metabolisme Macromoleculen

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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 requireenergy 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 powerneeded 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

http://www2.ufp.pt/~pedros/bq/glycolysis.htm


http://www2.ufp.pt/~pedros/bq/tca.htm


http://www2.ufp.pt/~pedros/bq/respi.htm


http://www2.ufp.pt/~pedros/bq/ppp.htm


http://www2.ufp.pt/~pedros/bq/urea.htm


http://www2.ufp.pt/~pedros/bq/fatty.htm




http://www2.ufp.pt/~pedros/bq/gng.htm











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 thephosphofrutokinase 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.

http://www2.ufp.pt/~pedros/bq/integration.htm#perfil


http://www2.ufp.pt/~pedros/bq/integration.htm#glucagon























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 areplenty 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










http://www2.ufp.pt/~pedros/bq/glycogen.htm

















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.







http://www2.ufp.pt/~pedros/bq/glycogen.htm#degrada





http://www2.ufp.pt/~pedros/bq/urea.htm#GlnS












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, anddepresses 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 least160 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.




http://www2.ufp.pt/~pedros/qo2000/aldeidos.htm#aldol







Phosphorilated acids (as well as phosphoenols and phosphoguanidines) contain very energetic

phosphate groups: hydrolysis of these phosphate groups yields with very significant resonancestabilization. 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-

http://www2.ufp.pt/~pedros/qo2000/resonancia.htm




http://www2.ufp.pt/~pedros/bq/glycolysis.htm#phosphato



http://www2.ufp.pt/~pedros/bq/glycolysis.htm#nadh














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 andphosphoenolpyruvate(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 tomalate in the mithochondrial matrix. The mithochondrial membrane is permeable to malate,




http://www2.ufp.pt/~pedros/bq/urea.htm#ceto







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 reverseof 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 forsurvival, 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 pyruvatedehydrogenase 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 (E

0= 0 mV)

as electron acceptor. Fumarate hydration yields malate, which can be oxidized tooxaloacetate, 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 -

http://www2.ufp.pt/~pedros/anim/2frame-iien.htm



http://www2.ufp.pt/~pedros/qo2000/alcanos.htm#hidrata















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 byglutamate-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

http://www2.ufp.pt/~pedros/bq/tca.htm#ceto




http://www2.ufp.pt/~pedros/bq/glycogen.htm#gpi















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 AMPcan 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 produceNADH 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.

http://www2.ufp.pt/~pedros/bq/tca.htm#succinato



http://www2.ufp.pt/~pedros/bq/urea.htm#asp







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. Inthese 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) :

http://www2.ufp.pt/~pedros/bq/urea.htm#desamina%C3%A7%C3%A3o














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 tostart 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 canonly 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 branchto 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.

http://www2.ufp.pt/~pedros/bq/urea.htm#b6






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 themuscle 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.

http://www2.ufp.pt/~pedros/bq/glycolysis.htm#pga






http://www2.ufp.pt/~pedros/bq/tca.htm#citrato








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, Δ

2-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, Δ

2system, 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 fattyacids 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 oldpatients 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 chainfatty 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 biotinas 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).




http://www2.ufp.pt/~pedros/bq/gng.htm#carbox


http://www2.ufp.pt/~pedros/bq/fatty.htm#impar








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-carbonacyl-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 synthesismust be produced by the pentose phosphate pathway.




http://www2.ufp.pt/~pedros/bq/fatty.htm#malica











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 betweenboth 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). Manymicroorganisms use SO42-

, SeO42-

,NO3-, NO2

-, NO, U

6+ (uranium), Fe

3+, 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 toubiquinol (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

http://ajpregu.physiology.org/cgi/reprint/287/3/R502





http://www2.ufp.pt/~pedros/anim/2frame-ien.htm












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 twosolutions 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 normalconditions, 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 theelectron transport chain. These are:

http://www2.ufp.pt/~pedros/anim/2frame-iiien.htm



http://www2.ufp.pt/~pedros/anim/2frame-iven.htm







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 producesalmost 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.

Overzicht metabolisme Macromoleculen

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