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You are what you eat

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Core Metabolism

Contents

• Glycolysis
• Pentose phosphate pathway
• Krebs cycle
• β-Oxidation
• Diagrams

Glycolysis

Glycolysis is of central importance to the metabolism of eukaryotic cells. It links the metabolism of sugars to that of organic acids in the Krebs cycle, and in anaerobic organisms, provides the principle route of energy (ATP) generation. The reactions are rather complex, but can be seen as four basic processes:

1. Isomerisation (catalysed by an isomerase): the intramolecular rearrangement of a molecule. This may be the transfer of a carboxyl group (C=O) from the end of a molecule (such as an aldose) to the middle (such as a ketose), or it may be the transfer of a phosphate group (the enzymes catalysing this latter sort of isomerisation are generally termed mutases).
2. Phosphorylation (catalysed by kinases): the movement of a phosphate group from one molecule (such as a phosphorylated sugar) to another (such as ATP).
3. Dehydration (catalysed by a dehydratase or hydrolase acting in reverse): the removal of water from a molecule.
4. Aldol cleavage (catalysed by an aldolase): the splitting of the carbon-carbon bond in a -(C=O)-CH(OH)-CH(OH)- molecule to generate a free aldehyde.

Many of these reactions are freely reversible, so the glycolytic pathway can (for the most part) be run in either direction. The formation of glucose from substrates lower in the pathway is termed gluconeogenesis, and follows the same biochemical pathway, give or take three steps (hexokinase, PFK and PEP kinase, see the diagram for these exceptions).

The entry of the various naturally occurring sugars into glycolysis takes place by a variety of different means, but most result in the formation of glucose- or fructose-6-phosphate. Glucose itself is phosphorylated to glucose-1-phosphate by hexokinase at the expense of ATP, and rapidly converted to glucose-6-phosphate by phosphoglucomutase. Fructose is phosphorylated to fructose-6-phosphate by fructokinase at the expense of ATP. Sucrose enters the pathways by cleavage by invertase to form glucose and fructose. Starch and glycogen are directly converted to glucose-6-phosphate by a phosphorylase using inorganic phosphate (reflecting the energy stored in the glycosidic bond). Other sugars enter by conversion to glucose phosphates, often by rather involved means: for example, galactose requires phosphorylation by galactokinase, followed by uridylation via a transferase, followed by epimerisation to glucose-1-phosphate.

Anyway, the upshot is that we get a pool of glucose-6-phosphate. This may be isomerised to fructose-6-phosphate by hexose phosphate isomerase. This is essentially the movement of the carboxyl group from C1 of glucose to C2 of fructose, and converts the aldose to a ketose.

The next step is one of the most important. Fructose-6-phosphate is phosphorylated to fructose-1,6-bis-phosphate by phosphofructokinase (PFK) at the expense of another molecule of ATP. This step is essentially irreversible, and is heavily regulated by allosteric means by ATP and many other metabolites. The conversion of FbP back to fructose-6-phosphate in gluconeogenesis is catalysed by a different enzyme (fructose-bis-phosphatase), which does not regenerate ATP, but merely cleaves off the phosphate group. This reflects the fact that the glycolytic pathway's equilibrium position lies far to the side of pyruvate, hence ATP can only be generated in the glucose → pyruvate direction. The hexokinase step is also irreversible, and a 'phosphate-wasting' enzyme (glucose-6-phosphatase) is required to generate glucose from is phosphate.

The product of PFK is fructose-1,6-bis-phosphate, which is unstable, and readily cleaved by an aldolase to form dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). These two triose sugars are isomeric, and are interconvertable by triose phosphate isomerase. The equilibrium lies well over to the side of DHAP, but glycolysis proceeds via GAP. The removal of GAP drives conversion of DHAP to GAP by Le Chatelier's principle.

So far, glycolysis has cost us ATP (one for the PFK reaction, and maybe another from a kinase, depending on whether the substrate was glucose or starch). The next few steps finally start to recover this ATP. Note that because the aldolase reaction produces two trioses, the remainder of the pathway is 'doubled up' with respect to the original hexose. The first step of these ATP-generating reactions is the oxidation and phosphorylation of GAP to 1,3-bis-phosphoglycerate (BPG) by GAP dehydrogenase. This reaction both adds a phosphate group to GAP (from inorganic phosphate), and abstracts two hydrogen atoms, which are attached to NAD to form NADH. NADH (nicotinamide adenine dinucleotide) is the cofactor that participates in most catabolic redox reactions. Its molecular sibling, NADP (nicotinamide adenine dinucleotide phosphate) serves the same function in most biosynthetic anabolic pathways, and is produced by the pentose phosphate pathway. Remember: NADH is for glycolysis/Krebs and NADPH is for pentose-phosphate/photosynthesis.

The next step finally starts reaping back our ATP investment. BPG is converted to 3-phosphoglycerate, generating ATP from ADP. This covers our initial costs, since this reaction occurs twice, once for each triose produced from glucose, and we break even on the two ATP required to generate fructose-1,6-bis-phosphate from glucose. The last two reactions have been a little involved, but essentially, they fulfil three roles: they generate one molecule of ATP, and one molecule of NADH, and convert a sugar into a carboxylic acid (an oxidation).

The final three steps of glycolysis generate a little more ATP. 3-phosphoglycerate is isomerised to 2-phosphoglycerate by a mutase enzyme. 2-phosphoglycerate is dehydrated by an enzyme called enolase, which converts the diol group (C(OH)-C(OH)) of 2-PGA to the alkene group (C=C) of phosphoenolpyruvate (PEP), with loss of water. Enols are an unstable tautomeric form that ketone may adopt (q.v. bases in DNA), and readily convert to their corresponding ketones. In the case of PEP, this is pyruvate: however, this also requires the loss of the phosphate group. 'Cleverly', this spontaneous loss of phosphate is coupled to ATP synthesis (a 'substrate level phosphorylation'): the phosphate group, rather than being lost, is transferred to ADP. The enzyme catalysing this reaction is (confusingly) called pyruvate kinase. I say confusingly, as this name is the reverse of the reaction just described, and the equilibrium lies far towards pyruvate. In fact, this is the third irreversible step in glycolysis, and its reversal in gluconeogenesis requires another enzyme: PEP carboxylase, which generates PEP from the Krebs intermediate oxaloacetate (bypassing the steps between PEP and acetyl Co-A, which we will discuss presently).

So, we have generated two (or three, if we started with starch) ATP profits, but we have also generated two molecules of NADH and two of pyruvate. This NADH must be oxidised back to NAD, or glycolysis will rapidly run to a halt.

In aerobic metabolism, the pyruvate is transferred to the mitochondria, where Krebs cycle works its magic, extracting even more energy from the carbon skeleton. The NADH is also oxidised here at the expense of molecular oxygen by the process of oxidative phosphorylation.

However, before eukaryotes had mitochondria, and whenever they lack oxygen, they fall back on a cheaper and less efficient process. Pyruvate is reduced at the expense of NADH, which regenerates the NAD, but also wastes the energy that could be extracted from pyruvate by further oxidation. This is termed anaerobic respiration, or fermentation (strictly, the whole glycolytic pathway is anaerobic, but it is these terminal reactions that mark out the respiration as anaerobic, rather than a prelude to aerobic metabolism by Krebs and oxidative phosphorylation).

Plants/fungi and animal have different routes for this. Plants and (infamously) yeasts and other fungi, convert pyruvate to acetaldehyde and carbon dioxide using pyruvate decarboxylase (essentially irreversible); the acetaldehyde is then reduced to ethanol (alcohol) by alcohol dehydrogenase (the same enzyme that is used to degrade ethanol in the mammalian liver).

In animals, particularly in mammalian muscle under oxygen deficit, the pyruvate takes a different route: it is converted in a single step to lactate, by lactate dehydrogenase. It is the accumulation of this lactate that causes some of the pain felt during exercise. This reaction is reversible, and can be run backwards to feed the lactate back to glycolysis/Krebs when oxygen becomes readily available again. It is important to note that both of these routes consume the same amount of NADH as was produced by glycolysis, so there is no net NAD consumption (hence glycolysis does not run to a halt).

So in summary, the central pathway of eukaryotic metabolism leads from glucose to pyruvate. Glucose is repeatedly phosphorylated, eventually yielding fructose-1,6bis-phosphate, which cleaves to form dihydroxyacetone phosphate and glyceraldehyde phosphate. These are both then stripped of phosphate group (with a small ATP profit), to yield pyruvate and NADH. The NADH is metabolically expensive, so it must be regenerated back to NAD⁺. Under anaerobic conditions, this is done by reacting it with pyruvate to yield reduced waste products, either two moles of lactate (animals, reversible), or two moles each of ethanol and carbon dioxide (plants and fungi, irreversible). Under aerobic conditions, NADH is fed to oxidative phosphorylation, and the pyruvate is further degraded to carbon dioxide by Krebs cycle. The yield of ATP from glycolysis is about 2 per glucose or 3 per starch-unit.

Pentose phosphate pathway

Glycolysis is not the only sugar-manipulating pathway in cells. DNA and RNA are both reliant on the production of the 5-carbon sugar ribose, and this is a central chemical in another oxidative pathway called the pentose phosphate pathway (PPP). The PPP is a network of reactions (as opposed to the largely straight-through pathway of glycolysis). This makes it a little difficult to understand, because it is capable of doing a great many things depending on how the individual reactions are tuned. The pathway generates a variety of sugars (including ribose), and unlike glycolysis, also produces NADPH, which is required by most cells for biosynthetic reactions. The pathway can be fiddled with by enzyme regulation to produce different ratios of NADPH to ribose.

There are four essential reactions in the PPP:

1. Oxidation: the initial step of PPP oxidises glucose to 6-phosphogluconate, with concomitant production of NADPH.
2. Decarboxylation: 6-phosphogluconate is then decarboxylated to ribulose-5-phosphate, producing another molecule of NADPH, and releasing CO₂.
3. Epimerisation: ribulose-5-phosphate is an epimer of ribose-5-phosphate and may be converted to it by a simple isomerisation proceeding by an enediol intermediate. This reaction is the same as that catalysing conversion of DHAP and GAP, and glucose- and fructose-6-phosphate in glycolysis, and is catalysed by a similar enzyme.
4. Transfer (transketolase and transaldolase reactions): most cells need far more NADPH than ribose. Consequently, the rest of the PPP consists of a variety of reactions which transfer 2-carbon (transketolase) and 3-carbon (transaldolase) units from sugar to sugar, generating a plethora of trioses, tetroses, pentoses, and even septuloses (epimerisation is also involved in this sequence). These reactions serve to regenerate fructose-6-phosphate and GAP, which may either be fed into glycolysis, or used to regenerate glucose-6-phosphate by gluconeogenesis.

Depending on the relative importances of NADPH and ribose synthesis, the PPP can be tuned to:

• Generate masses of ribose (dividing cells need this for DNA synthesis):

  5 Glucose-6-P + ATP → 6 Ribose-5-P + ADP

• Generate NADPH and ribose in balanced quantities:

  Glucose-6-P + 2 NADP + H₂O → Ribose-5-P + 2 NADPH + CO₂

• Generate masses of NADPH (adipose tissue needs this for the synthesis of fatty acids) and simultaneously oxidise glucose completely:

  Glucose-6-P + 12 NADP + 7 H₂O → 12 NADPH + 6 CO₂ + P_i

• Any stoichiometry in between these extremes.

The PPP is very important in plants, where is comprises part of the Calvin cycle, which regenerates ribulose-5-phosphate from its carboxylation products. The PPP is often overlooked in biology, but it is of at least as much importance as glycolysis, and may well have evolved before it.

Krebs cycle

The Krebs cycle (which in eukaryotes, occurs exclusively in the mitochondrial matrix) serves to take pyruvate (C₃H₃O₃) apart, smashing it into carbon dioxide and hydrogen 'atoms' (which are attached to nucleotide cofactors). The energy release by this is captured in GTP. The reason it is termed a cycle is because the raw material (C2 acetyl groups from pyruvate) are combined with (C4) oxaloacetate, to form a C6 acid (citrate), which is successively decarboxylated to regenerate the oxaloacetate we started with.

The initial step of the Krebs cycle (or tricarboxylic acid, or citric acid cycle), starts where glycolysis leaves off, with pyruvate. The first reaction of the Krebs cycle is to decarboxylate it. This is achieved by a very large (getting on for ribosome-sized) enzyme complex called pyruvate dehydrogenase. This enzyme abstracts carbon dioxide from pyruvate, with concomitant production of NADH and an acetyl group, which is attached to cofactor-A (a cofactor comprised of adenine and pantothenic acid - vitamin B₅).

Acetyl-CoA is then condensed to oxaloacetate to generate the tricarboxylic acid, citrate, by citrate synthase, which is then isomerised to isocitrate via aconitate by the enzyme aconitase.

Isocitrate is then subjected to a similar reaction to pyruvate: a simultaneous dehydrogenation and decarboxylation by isocitrate dehydrogenase, via the short lived intermediate oxalosuccinate to form α-ketoglutarate (2-oxoglutarate). This is the second step generating CO₂ in the destruction of pyruvate, so if you're keeping count, we only have one more carbon to go.

α-ketoglutarate (2-oxoglutarate), a 5-carbon acid, is dehydrogenated and decarboxylated to succinyl coenzyme-A by α-ketoglutarate dehydrogenase, which is a similarly complicated system as that generating acetyl-CoA. This removes the last carbon atom added by acetyl-CoA to the cycle, so the remainder of the cycle exists to convert this 4-carbon acylated cofactor back to oxaloacetate.

The cleavage of the thioester bond in succinyl CoA is coupled to the phosphorylation of GDP to GTP. GTP is much like ATP, and is readily interconverted with ADP to ATP and GDP by nucleoside diphosphokinase. This reaction is catalysed by the (confusingly named) succinyl CoA synthetase, and is the only Krebs cycle reaction that produces substrate-level phosphorylation.

The succinate produced by succinyl-CoA cleavage is then dehydrogenated by succinate dehydrogenase to fumarate, a membrane-bound enzyme also known as Complex II, since it feeds directly into the process of oxidative phosphorylation. In books, this is often written as FAD → FADH₂, although the flavin cofactor is only transiently oxidised, and is an integral part of the enzyme, not a freely diffusable cofactor like NADH.

Fumarate is then hydrated stereospecifically to L-malate by the addition of water across its double bond. This is catalysed by the enzyme fumarase. The malate thereby formed is dehydrogenated by malate dehydrogenase to regenerate oxaloacetate.

The net reaction of the Krebs cycle is:

Acetyl-CoA + 3 NAD + FAD + GDP + P_i + 2 H₂O → 2 CO₂ + 3 NADH + FADH₂ + GTP + 2 H⁺ + CoA

The main thing to note is the production of large quantities of reduced cofactors. These can only be oxidised by the the process of oxidative phosphorylation, hence the Krebs cycle only runs under aerobic conditions, even though none of its reactions have a direct requirement for molecular oxygen.

As well as being used to degrade pyruvate, and therefore glucose, the Krebs cycle is also essential for amino acid synthesis and catabolism. In particular α-ketoglutarate and oxaloacetate are used to generate many amino acids by transamination. The Krebs cycle in fact was the second Krebs cycle, since the first he discovered (with Henseleit) was the urea cycle. This shares two intermediates with the Krebs (citrate) cycle (oxaloacetate and fumarate), and is used to generate urea, via a variety of amino acid intermediates.

Because intermediates are removed from the cycle for these processes, the amount of oxaloacetate is reduced, and consequently, the cycle will grind to a halt, as acetyl-CoA cannot eneter without oxaloacetate to condense with. Consequently, oxaloacetate and other intermediates are added to the cycle by anaplerotic reactions, such as pyruvate carboxylase, which generates oxaloacetate from pyruvate and CO₂ at the expense of ATP. These anaplerotic reactions serve to keep the cycle topped up. Plants and bacteria have a different modification: they can use a shortcut through the cycle via glyoxylate which is produced along with succinate from isocitrate by isocitrate lyase. Acetylation (from acetyl-CoA) of glyoxylate by malate synthase regenerates malate. This 'glyoxylate cycle' is essentially a way of sticking two acetyl groups together to form succinate, which can be used in biosynthesis.

In summary, the Krebs cycle occurs exclusively in aerobic mitochondria, generating carbon dioxide, NADH, ATP and FADH₂. Pyruvate is fed into the cycle as acetyl-coenzyme A. The enzyme converting pyruvate to acetyl-CoA is pyruvate decarboxylase: it removes one of pyruvate's three carbons as carbon dioxide. ACoA combines with oxaloacetate produced by Krebs cycle to produce citrate. By successive oxidation reactions (via ketoglutarate, succinate and malate), two moles of carbon dioxide are released from the remains of the ACoA, regenerating oxaloacetate, and producing reduced nucleotides (mostly NADH), which are fed to oxidative phosphorylation.

β-Oxidation

Fatty acid metabolism also feeds into Krebs and/or glyoxylate cycles. There are several ways to degrade fatty acids, but the commonest is β-oxidation.

In β-oxidation, fatty acids are degraded by means similar to that used to degrade pyruvate and isocitrate, in that the fatty acid is linked to coenzyme-A before it is metabolised. This acyl Co-A is then subjected to a repeated series of reactions, which serve to cleave the acyl chain into two components: an acetyl-CoA unit, and a 'new' acyl-CoA unit that has been shortened by two units.

1. Triacylglycerols are hydrolysed by lipase to generate glycerol (not shown) and a long-chain fatty acid.
2. The long chain fatty acid will be degraded from the COOH end.
3. The fatty acid is linked to CoA by acyl-CoA synthetase, at the expense of ATP
4. The fatty acid is oxidised by acyl-CoA dehydrogenase, which abstracts hydrogen onto FAD, and thence to oxidative phosphorylation. The product is a trans-Δ²-enoyl CoA, which is then hydrated by enoyl-CoA hydratase.
5. This generates a 3-hydroxyacyl-CoA, which is further oxidised by 3-hydroxyacyl-CoA dehydrogenase, producing NADH.
6. This generates a ketone, ketoacyl-CoA.
7. This ketone is then cleaved by an enzyme called thiolase, which uses coenzyme-A to generate a shorter acyl-CoA, and an acetyl unit (linked to the original coenzyme-A).

The acetyl coenzyme A units produced by β-oxidation then feed into Krebs cycle in the same way as pyruvate. In germinating plant seeds, specialised glyoxysomes use the glyoxylate cycle to generate succinate for amino acid biosynthesis and gluconeogenesis, from stored fat reserves. Animals are incapable of doing this, because they lack the glyoxylate short-cut. Consequently, they cannot make glucose from fat, since they cannot feed acetyl-CoA into a anaplerotic reaction.

It is interesting to note that chemically, these steps are very similar to those of the last portion of the Krebs cycle: an acyl group (succinate) is converted to an enoyl group (fumarate), which is converted to a hydroxyacyl group (malate), which is converted to a keto group (oxaloacetate).

Diagrams

You may find these five diagrams useful:

• Glycolysis.
• Krebs cycle.
• Urea cycle.
• β-oxidation.
• Pentose phosphate pathway.