METABOLIC PATHWAYS

GLYCOLYSIS:

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 inglycogen synthesis(a storage form of glucose) , production of other carbon compounds by thepentose-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 byphosphofructokinase. This is thecommited stepof 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 preventsgluconeogenesisfrom 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 inversealdolic additioncleaves fructose-1,6-bisphosphate intwo 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.

Aldehydeshave 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 significantresonance 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-Phosphoglycerateis isomerized to 2-phosphoglycerate, which after dehydration (i.e. losing H2O) yields a phosphoenol:

Dueto itshigh phosphate transfer potentialphosphoenolpyruvate 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 inone of the reactions. Under aerobic conditions, NADH transfers its two electrons to theelectron-transport chain. In animal cells, in the absence of O2NADH transfers its electrons to the end-product of glycolysis (pyruvate), yielding lactate. This is calledfermentation: an internally balanced degradation, i.e., a process that uses one of its products as the final acceptor of the electrons it releases.

CITRIC ACID CYCLEPyruvateproduced 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 thecitric acid cycle. First, pyruvate is decarboxylated toacetyl-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 andgluconeogenesis.

In the first reaction of the citric acid cycle, acetyl-CoA attacks oxaloacetate, yielding citrate, in analdol addition. Thioester hydrolysis helps to displace equilibrium towards product formation:

Citrate is then isomerized to isocitrate, which is then decarboxylated toa-ketoglutarate. If citrate had not been isomerized to isocitrate, this decarboxylation would yield a branched carbon compound, much harder to metabolize.

a-ketoglutarate is aa-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 (a-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 hydrolysiswill 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 thesuccinate 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. Fumaratehydrationyields malate, which can be oxidized to oxaloacetate, thus closing the cycle.A similar sequence of reactions happens infatty acidsb-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

GLUCONEOGENESIS

The human body has two main ways to keep constant blood glucose levels between meals:glycogen degradationandgluconeogenesis.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 ofglycolysis. 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 itsDG is rather positive. In order to overcome this thermodynamic barrier, the reaction will be coupled to adecarboxylation, 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 thecitric acid cycle. Since both pyruvate and phosphoenolpyruvate(PEP) are three-carbon compounds, pyruvate must becarboxylatedto 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 bynumaphosphoenolpyruvate 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 betransaminatedto 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).

Glycogen Synthesis and Degradation

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 inglycogen synthesis(a storage form of glucose) , production of other carbon compounds by thepentose-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 typea-1,4 anda-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 enzymefosfoglucomutase.

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 beactivated, i.e., transformed into a species with high phosphate transfer potential.This isaccomplished 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-glucosehas 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 calledglycogenin. 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 degradationGlycogen is degraded by the sequential action of three enzymes:

glycogen phosphorylasecleavesa(1-4) bonds with inorganig phosphate(Pi). It can only cleave glucose residues 4 (or more) glucose residues away from a branching point . It usespyridoxal, a vitamin B6derivative, 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 aa(1-6) glycosidic bond) is removed byhydrolysis, 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:

Pentose Phosphate PathwayIn 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 fromglycolysis, thepentose-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 producesribose-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 thenon-oxidativepart 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 byglycolysisin orer to produce energy, or recycled throughgluconeogenesisto 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 CO2molecules, 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.

Fatty Acid MetabolismFatty 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) todihydroxyacetone phosphate. Both electrons are taken up by ubiquinone (Q), and are fed into theelectron transport chain.

Fatty acids follow a different pathway:b-oxidation, which takes place in the mitochondrion. Before entering the mitochondrion, fatty acids must beactivated. 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 thecitric 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 carnitineis 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 acidsb-oxidation is a cyle composed of three consecutive reactions, which are identical tothe 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 enzymethiolasereleases 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, 3, 2-enoyl-CoA isomerase is needed: this enzyme transfers the double bond from C3 to C2, thereby allowing -oxidation. In this -oxidation cycle no FADH2is 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 4, 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 ofeven-chain fatty acidsin acetyl-CoA, which can becompletelyoxidized to CO2through thecitric acid cycle: even-chain fatty acids cannot be used fornetsynthesis of oxaloacetate, and therefore are not a substrate forgluconeogenesis.

In the last round ofb-oxidation,odd-chain fatty acidsyield 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 bycarboxilation. Methylmalonyl-CoA formed in this reaction is then rearranged succinyl-CoA, in a cobalamine (a vitamin B12derivative)-assisted reaction.

Succinyl-CoA is an intermediate in the citric acid cycle and also a precursor of heme biossynthesis. A vitamin B12deficiency therefore impairs the ability to synthesize heme and may eventually lead to the onset ofpernicious 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 B12stock enough for about 3-5 years, so that the effects of an impairment of its absorption will be very delayed.

Succinyl-CoA can beoxidized by the citric acid cycleto malate, which after moving into the cytoplasm can be used ingluconeogenesis. In the cytoplasm, malate can also be decarboxylated to pyruvate by themalic enzyme, with cincommitant NADPH production:

Pyruvate formed in this reaction can enter the mitochondrion and be oxidized completely to CO2by thecitric acid cycle.

Peroxisomal degradation of fatty acids

Peroxisomes are small organelles where the initial steps ofb-oxidation of very long chain fatty acids occur. The major differences between mitochondrial and peroxisomalb-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 acidb-oxidation in liver mitochodria is converted inacetoacetateandb-hydroxybutyrate(also known asketone 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 thecondensation 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, yieldingsuccinateand 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 ofb-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 (AcylCarrierProtein), instead of coenzyme A.

Fatty acids synthesis uses acetyl-CoA as main substrate. However, since the process is quite endergonic acetyl-CoA must beactivated, which happens throughcarboxylation. Like other carboxylases (e.g., those ofpyruvateorpropionyl-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 ofb-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 byelongasesanddesaturases.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 thecitrate shuttle: citrate is formed in the mitochondrion bycondensing acetyl-CoA with oxaloacetateand 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 themalic enzyme. The remainder of the NADPH needed for fatty acid synthesis must be produced by thepentose phosphate pathway.