Biochemistry: Carbohydrate Metabolism & Pentose Phosphate Pathway

• Anabolic pathway that utilises the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents.
• It generates reducing equivalents in the form of NADPH for reductive biosynthesis ractions within cells, provides the cell with ribose-5-phosphate for the synthesis of nucleotides and nucleic acids and it operates to metabolise dietary pentose sugars from the digestion of nucleic acids.

Overall Equation:

3G6P + 6NADP⁺ + 3H₂O ⇄ 6NADPH + 6H⁺ + 3CO₂ + 2F6P + GAP

There are 3 Phases: Oxidative, Isomerisation & epimerisation and C-C bond cleavge and formations. Reactions take Place in the Cytosol.

Phase 1 Yields NADPH and Ribulose-5-Phosphate (Ru5P)

3 G6P + 6NADP⁺ + 3H₂O → 6NADPH + 6H⁺ + 3CO₂ + 3Ru5P

The First 3 reactions produces 2 NADPH + H⁺ and eliminates 2CO₂

• Glucose-6-Phosphate dehydrogenase (G6PD) catayses the transfer of a hydride ion to NADP⁺ from Carbon 1 of Glucose-6-Phosphate to form 6-phosphogluconolactone.
• 6-Phosphogluconolactonase increases the rate of hydrolysis of  6-phosphogluconolactone to 6-phosphogluconate
• 6-Phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 6- Phosphogluconate, a beta hydroxy acid, to Ru5P and CO₂.

The formation of Ru5P completes Phase 1. It generates 2 NADPH for each G6P molecule.

Phases 2 & 3 are reversible so the products vary with the needs of the cell.

Isomerisation and Epimerisation reactions transform Ribulose-5-Phosphate (Ru5P) to Ribose-5-Phosphate (R5P) OR to Xylulose-5-Phosphate (Xu5P)

3Ru5P ⇌ R5P + 2Xu5P

• Ru5P is converted to R5P by ribulose-5-phosphate isomerase
• Ru5P is converted to Xu5P by ribulose-5-phosphate epimerase
• These Reactions occur via enediolate intermediates.

When the need for R5P is more than NADPH, F6P and GAP can be diverted from the glycolytic pathway for use in the synthesis of R5P by reversal of the transaldolase and transketolase reactions.

When the need for NADPH exceeds that of R5P The transaldolase and transketolase reactions convert excess R5P to glycolytic intermediates. GAP and F6P produced can be consumed through glycolysis and oxidative phosphorylation.

C-C bond cleavage and formation reactions convert 2 molecules of Xu5P and 1 molecule of R5P to 2 molecules of F6P and 1 molecule of GAP.

A series of reactions that convert three of the pentose-5-phosphates into two molecules of hexoses and one triose. Catalysed by two enzymes Transaldolase and Transketolase.

Transketolase (contains a thiamine pyrophosphate cofactor TPP) catalyses the transfer of a C₂ unit from Xu5P to R5P yielding GAP and sedoheptulose-7-phosphate S7P.

Transaldolase catalses the transfer of a C₃ unit from S7P to GAP yielding erythrose-4-phosphate (E4P) and F6P. This reaction occurs by aldol cleavage which begins with the formation of schiff base between a Lysine residue amino group and carbonyl group of S7P.

In a second transketolase reaction Glyceraldehyde-3-phosphate and another Fructose-6-phosphate molecule are produced by transferring a C₂ unit from Xu5P to E4P to form GAP and F6P.

Hexoses such as Fructose, Galactose and Mannose are converted into glycolytic intermediates in order to enter the glycolytic pathway for metabolism.

Fructose (muscle) and Mannose- F6P  Fructose (Liver)-GAP  Galactose-G6P

Fructose is Converted to Fructose-6-Phosphate or Glyceraldehyde-3-Phosphate

• Metabolism of Fructose occurs in the muscle and liver.Hexokinase phosphorylates fructose yielding F6P. Entry of fructose into glycolysis involves one reaction step. The liver converts fructose to glycolytic intermediates through a pathway:
• Fructokinase catalyses the phosphorylation of fructose by ATP at C1 to form fructose-1-phosphate. 
• The liver contains Type B aldolase for which F1P is also a substrate. Fructose-1-phosphate therefore undergoes an aldol cleavge:

Fructose-1-phosphate ⇌ dihydroxyacetone phosphate + glyceraldehyde

• Direct phosphorylation of Glyceraldehyde by ATP through the action of glyceraldehyde kinase forms the glycolytic intermediate GAP
• Alternatively, glyceraldehyde is converted to glycolytic intermidiate DHAP (Dihydroxyacetone phosphate) beginning with its NADH dpenedent reduction to glycerol catalysed by alchol dehyrogenase.
• Glycerol kinase catalyses ATP dependent phosphorylation to produce Glycerol-3-Phosphate.
• DHAP is produced by NAD⁺ dependent oxidation catalysed by Glycerol phosphate Dehydrogenase.
• The DHAP ris then converted to GAP by triose phosphate isomerase.

In the muscle Fructose conversion to F6P involves Hexokinase, In the liver seven enzymes participate in its conversion.

Galactose is Converted to Glucose-6-Phosphate

Galactose is obtained from lactose hydrolysis. An epimerisation reaction must occur before gaalctose eneters glycolysis. This happens after the conversion of galactose to its uridine diphosphate derivative. The conversion of Galactose to a glycolytic intermediate requires 4 reactions:

• Galactose is phosphorylated at C1 by ATP in a reaction catalysed by Galactokinase.
• Galactose-1-phosphate uridylyl transferase transfers the uridylyl group of UDP-glucose to galactose-1-phosphate to yield glucose-1-phosphate (G1P) and UDP-galactose by the reversible cleavage of UDP-glucose's pyrophosphoryl bond.
• UDP-galactose-4-epimerase converts UDP-galactose back to UDP-glucose. 
• G1P is converted to the glycolytic intermediate G6P by Phosphoglucomutase.

Mannose is converted to Fructose-6-Phosphate

Mannose (the C2 Epimer of glucose) enters the glycolytic pathway after its conversion to F6P via a 2 reaction pathway:

• Hexokinase recognises mannose and converts it to mannose-6-phosphate.
• Phosphomannose isomerase then converts this aldose to the glycolytic intermediate F6P in a reaction similar to phosphoglucose ismomerase.

• NADH, a substrate for the GAPDH reaction, must be reoxidized for glycolysis to continue.

• In muscle, pyruvate is reduced to lactate to regenerate NAD+

• Yeast decarboxylates pyruvate to produce CO2 and ethanol, in a process that requires the cofactor TPP.

3 Common Metabolic fates of pyruvate produced in glycolysis:

• Under aerobic conditions, the pyruvate is completely oxidised via the citric acid cycle to CO₂ and H₂O.
• Under anaerobic conditions, pyrivate must be converted to a reduced end products in order to reoxidise NADH produced by the GAPDH reaction, this can occur in two ways: 
• Under anerobic conditions in muscle pyruvate is reduced to lactate to regenrate NAD+ in a process known as homolactic fermentation. 
• In yeast pyruvate is decrabvoxylated to yield CO₂ and acetaldehyde which is then reduced by NADH to yield NAD+ and ethanol.- alcoholic fermentation.

In  muscle during vigrous activity when ATP demand is high and O2 is in short supply. ATP is synthesised via anaerobic glycolysis. Lactate dehydrogenase catalyses the oxidation of NADH by pyruvate to yield NAD+ and lactate. This is a freely reversible reaction.

The Overall process of anaerobic glycolysis in muscle can be represented as

Glucose + 2ADP + 2P_i → 2Lactate + 2ATP + 2H₂O + 2H+

Much of the lactate produced can be converted back to pyruvate or carried by blood to the liver to synthesise glucose. 

Yeast produces ethanol and CO₂ via two reactions:

• The decarboxylation of pyruvate to form acetaldehyde and CO₂  as catalysed by pyruvate decarboxylase.
• The reduction of acetaldehyde to ethanol by NADH as catalysed by alcohol dehydrogenase therby regenrating NAD+ for use in the GAPDH reaction of glycolysis.

TPP (thiamine pyrophosphate) is a cofactor of pyruvate decarboxylase it binds to it tightly and non covalently.

TPP stabilises the carbanion intermediates as an electrophile by non covalent bonds.

Yeast decarboxylates pyruvate to produce CO2 and ethanol, in a process that requires the cofactor TPP.

Several steps in glycolysis are regulated, but the most important control point is the third step of the pathway, which is catalyzed by an enzyme called phosphofructokinase (PFK). This reaction is the first committed step, making PFK a central target for regulation of the glycolysis pathway as a whole,

PFK is regulated by ATP, an ADP derivative called AMP, and citrate.

ATP is a negative regulator of PFK if there is already plenty of ATP in the cell, glycolysis does not need to make more.

AMP. Adenosine monophosphate (AMP) is a positive regulator of PFK. When a cell is very low on ATP, it will start squeezing more ATP out of ADP molecules by converting them to ATP and AMP High levels of AMP mean that the cell is starved for energy, and that glycolysis must run quickly to replenish ATP.

Citrate, the first product of the citric acid cycle, can also inhibit PFK. If citrate builds up, this is a sign that glycolysis can slow down, because the citric acid cycle is backed up and doesn’t need more fuel.

Glycolysis is a 10 reaction sequence involving the breakdown of glucose to  2 pyruvate (C3 units) while using the free energy rleased to synthesise ATP from ADP and P_i.Glycolyisis can be divded into two stages:

• Energy investment- the hexose glucose is phosphorylated and cleaved to yield two molecules of Glyceraldehyde-3-phosphate. This consumes 2 ATP
• Energy recovery- The two molecules of glyceraldehyde-3-phosphate are converted to pyruvate with generation of 4 ATP. Theres net profit of 2ATP per glucose molecule. 
• NaDH is continually reoxidised during glycolysis to keep the pathway supplied with energy and  a primary oxidising agent NAD+

The overall Glycolysis Reaction is:

Glucose + 2NAD⁺ + 2ADP + 2P_i → 2 Pyruvate + 2NADH + 2ATP +2H₂O + 4H⁺ 

Reaction 1: Transfer of a phosphoryl group from ATP to glucose to form Glucose-6-Phosphate (G6P) in a reaction catalysed by hexokinase.

Reaction 2: Glucose-6-Phosphate is converted to Fructose-6-Phosphate (F6P) by phosphoglucose isomerase (PGI)

• This is the isomerisation of an aldose to a ketose. the reaction mechanism involes genral acid base catalysis by the enzyme- the substrate binds then an enzymatic acid (the amiono group of a conserved Lys residue) catalyses the ring opening.
• A base (His imidazole group) abstracts the acidic proton from C2 to form a cis-enediolate intermediate.
• G6P is converted to F6P by intramolecular proton transfer before the 3H has has a chance to exchange with the medium
• The ring closes to form the product which is released to yield free enzyme, therby completing the catalytic cycle.

Reaction 3: Phosphofructokinase (PFK) phosphorylates F6P to yield Fructose-1,6-biphosphate.

The enzyme catalyses the nucleophillic attack by the C1-OH group of F6P on the electrophillic phosphorous atom of the Mg ²⁺ -ATP complex.

Reaction 4: Alsolase catalyses the cleavage of Fructose-1,6-biphosphate (FBP) to form the two trisoes Glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)

• The substrate FBP binds to the enzyme
• The FBP carbonyl group reacts with amino group of active site Lys to form an iminium cation
• The C3-C4 bond is cleaved forming an enamine intermediate and relasing GAP. Catalysis occurs because the enamine intermediate is more stable than the enolate intermediate of the base catalysed aldol cleavage reaction.
• Protonation and tautomerisation of the enamine yield the iminium cation from the schiff base.
• Hydrolysis of the iminium cation releases DHAP and regenerates the free enyme.

Reaction 5: GAP continues along the Glycolytic pathway. DHAP and GAP are interconverted by an isomerisation reaction with an enediol intermediate. Triose phosphate isomerase catalyses this process. At this point 1 molecule of glucose has been transfomed into 2 molecules of GAP. 2 ATP has been consumed in generating phosphorylated intermediates.

Stage 1- Hexose is Phosphorylated, Isomerised, Phosphorylated then Cleaved to GAP and DHAP.

Reaction 6: The oxidation and phosphorylation of GAP by NAD+ and P_i  catalysed by Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to produce 1,3-Bisphosphoglycerate (1,3-BPG)

• GAP binds to the enxyme 
• The essential sulfhydryl group, acting as a nucleophile, attacks the aldehyde to form a thiohemiacetal
• The thiohemiacetal undergoes oxidation to an acyl thioester by direct hydride transfer to NAD+. This intermediate has a large free energy of hydrolysis.
• P_i binds to the enzyme-thioester-NADH complex
• The thioester intermediate undergoes nucleophillic attack by P_i to form the high energy mixed anhydride, 1,3-BPG product which then dissociates from the enzyme followed by replacement of NADH by another molecule of NAD+ to regenerate the active enzyme.

Reaction 7: From 1,3-BPG Yields ATP together with 3-Phosphoglycerate (3PG) in a reaction catalysed by Phosphoglycerate kinase (PGK)

Reaction 8: 3PG is converted to 2-Phosphoglycerate (2PG) by phosphoglycerate mutase (PGM)

Reaction 9: 2PG is dehydrated to Phosphoenolpyruvate (PEP) in a reaction catalysed by enolase.

Reaction 10: Pyruvate Kinase couples the free energy of PEP cleavge to the synthesis of ATP during the formation of pyruvate

• A Phosphoryl oxygen of ADP nucleophillically attacks the PEP phosphorus atom displacing enolpyruvate and forming ATP
• Enolpyruvate tautomerizes to pyruvate.

In Stage 2 of Glycolysis 2 phosphorylated C3 units are transformed to two pyruvates with the synthesis of 4ATP. 3 products are: ATP, NADH and pyruvate.