3 MAIN STAGES/PARTS of GLYCOLYSIS

Stage 1Conversion of glucose into fructose 1,6-

bisphosphate3 STEPS

1. Phophorylation 2. Isomerization

3. 2nd PhosphorylationFor what?

- To trap glucose in the cell and form a compound that can readily be cleaved into

phosphorylated 3-carbon units

Stage 2Cleavage of Fructose 1,6-bisphosphate into

two 3-carbon fragments

Stage 3Harvesting of ATP when 3-carbon fragments

are oxidized to pyruvate

GLYCOLYSIS (Embden-Meyerhof Pathway) principal route for glucose metabolism main pathway for the metabolism of fructose,

galactose, and other carbohydrates derived from the diet

main Pathway for Glucose Oxidation used by all cells to extract energy from glucose all enzymes are in the cytosol generates ATP (substrate level phosphrylation:

only 2 ATPs) oxidation of glucose yields up to 38 ATPs in

Aerobic Conditions but only 2 ATP when O2 is absent

end product – pyruvate for complete oxidation of pyruvate, the following

are needed:

1. molecular oxygen

2. mitochonfrial enzymes

- pyruvate dehydrogenase complex

- citric acid

- respiratory chain

2 Phases of Glycolsis:

1. Preparatory or investment phase- from phosphorylation of glucose to splitting into 2 triose phosphates

2. Payoff phase- conversion of glyceraldehyde 3-phosphate to pyruvate with ATP formation

Overall Equation:

Glucose + 2 ADP + 2Pi 2 Lactate + 2 ATP + 2 H20

• Has two types: Aerobic (in presence of oxidation) and Anaerobic (e.g. erythrocytes)

Anaerobic glycolysis- erythocytes do not have mitochondria;

they metabolize glucose thru the anaerobic pathway

allows skeletal muscles to perform at very high levels when oxygen supply is inefficient, and it allows tissues to survive anoxic episodes

glycogen disappears and lactate appears

limits the amount of ATP formed per mole of glucose oxidized. Therefore, more glucose must be metabolized

when oxygen is admitted, aerobic recovery takes place and lactate disappears

erythrocytes metabolize glucose anaerobically as they do not have mitochondria

Anaerobic Degradation of Glucose to Lactate

-forms lactic acid or lactate-6-Carbon Glucose will yield 2

Pyruvate, which will then yield 2 Lactate

D-Glu 2 Pyruvate 2 Lactate

Product: 4 ATPs in GLYCOLYSIS minus (–) 2 ATPs used = 2 ATP TOTAL

Aerobic Glycolysis-converts Glucose to Pyruvate in the presence

of Oxygen (Product: Pyruvate)-sets the stage for complete oxidation of Glucose to CO2 and H2O -if Lactate is formed (from Pyruvate) which is

temporary, it is converted back to Pyruvate-Pyruvate is then converted to Acetyl CoA to

enter citric acid cycle

1 Glucose 2 Pyruvate Acetyl CoA

Enzyme used: NAD = 3 ATPs produced from conversion of Pyruvate to Acetyl CoA

**Importance of Formation of Pyruvate / Oxidation of Glucose

-can produce more ATP if Glucose is oxidized by O2 because it will pass the ETC

-oxidation of glucose can produce as much as 38 ATPs

-if it is unoxidized, ATP production will be very small (only 2-ATPs)

Table 1: 3 Main Stages of Glycolysis

PYRUVATE AEROBIC- CO2 + H2O through Citric Cycle

and Electron Transport Chain ANAEROBIC- lactic acid or ethanol

- less energy is generated

Lactic Acid - in skeletal muscle, when energy needs outspace the ability to transport oxygen

Why use Fermentation/Anaerobic?- oxygen is not needed to create

ATP

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RELATIONSHIP BETWEEN LACTATE AND PYRUVATE Pyruvate is the end product of aerobic glycolysis

as noted earlier. In the end of anaerobic glycolysis, lactate is formed from pyruvate. This reaction takes place in the cells of higher organisms when the amount of oxygen is limiting, as in muscle during intense activity.

The reduction of pyruvate by NADH to form lactate is catalyzed by LACTATE DEHYDROHENASE.

The regeneration of NAD+ in the reduction of pyruvate to lactate sustains the continued operation of glycolysis under anaerobic conditions.

REACTIONS OF GLYCOLYSIS

STEP 1 (IRREVERSIBLE)

Glucose + ATP glucose 6-phosphate + ADP + H

HEXOKINASE OR GLUCOKINASE converts glucose into glucose 6-phosphate through PHOSPHORYLATION

Hexokinase is found in all cells Glucokinase is found in the liver Glucokinase is activated when cells within the

body are saturated with glucose

Hexokinase and glucokinase are irreversible steps, and produce G-6-P, which is trapped in the cell.

- 1 ATP is consumed during this reaction ; Requires mg2+

- Reaction is irreversible

Comparison between Hexokinase and Glucokinase

Hexokinse GlucokinaseOccurrence in all tissues only in liverKm value 10-2 mmol/L 20 mmol/LAffinity for substrate

high low

Specificityacts on:

glucose;fructose

Acts on: mannose; glucose

Induction not inducedinduced by insulin and

glucose

Function

glucose is utilized by cells even if

blood sugar level is low

acts only when blood

glucose level is more

than 100mg/dl;glucose then is taken up by liver cells for

glycogen synthesis

STEP 2 (REVERSIBLE)• specific for glucose 6-phosphate and fructose 6-

PO4

Glucose 6-Phosphate + ATP ↔ Fructose 6-Phosphate

MUTASE/ISOMERASE is the enzyme responsible for changing the configuration

G6P is converted to F6P through ISOMERIZATION

Convert aldolase/pyranose to ketose/furanose

STEP 3 (IRREVERSIBLE)

Fructose 6-phosphate + ATP Fructose 1,6- bisphosphate +ADP + H

PHOSPHOFRUCTOKINASE (PFK), an allosteric enzyme that sets the pace of glycolysis

PHOSPHORYLATION of ATP to form another monophosphate which is attached to C1 of the fructose

the prefix bis in bisphosphate means two seperate monophosphate groups are present

- inhibited by ATP; stimulated by increase in ADP and AMP

- utilizes 1 ATP as the source of the phosphate transferred to fructose

STEP 4

Fructose 1,6-bisphosphate ↔ dihydroxyacetone phophate (DHAP) + glyceraldehyde 3- phosphate (GAP)

ALDOLASE is the enzyme used to form the 3 carbon fragments.

o Aldolase splites F-1,6-bis-P into two

seperate molecules, glyceraldehyde-3-P and dihydroxyacetone phosphate (DHAP). Only glyceraldehyde-3-phosphate can continue in glycolysis.

reversible but has a strongly positive standard free-energy change in the direction of cleavage

CLEAVAGE of the 6-carbon molecule triose phosphates are removed quickly by the

next 2 steps pulling the reaction in the direction of cleavage

STEP 5

Dihydroxyacetone phosphate (DHAP) ↔ glyceraldehyde 3-phosphate (G3P)

Enzyme TPI (TRIOSE PHOSPHATE ISOMERASE) is the enzyme that allows the conversion of DHAP to G3P.

G3P proceeds on the direct pathway of glycolysis, DHAP does NOT. But TPI is there to convert DHAP to G3P through ISOMERIZATION

Although the reaction is reversible, the reaction proceeds readily in forming G3P from DHAP.

TPI catalyzes the transfer of a hydrogen atom in converting DHAP into G3P which is an OXIDATION-REDUCTION reaction.

Everthing occurs in duplicate after this step occurs

• C1,C2 and C3 of glucose becomes Dihydroxyacetone phosphate

• C4,C5 and C6 becomes glyceraldehyde phosphate

STEP 6

NOTE: o At this point, glycolysis has transformed one

molecule of glucose into 2 molecules of glyceraldehyde 3-phosphate

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o HARVEST TIME!!!

Glyceraldehyde 3-phosphate + Pi + NAD+ ↔ 1,3- bisphosphoglycerate + NADH

GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE converts glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate, by PHOSPHORYLATION COUPLED TO OXIDATION

• glyceraldehyde-3-phosphate dehydrogenase- using NAD+ as a cofactor and generating NADH worth 3 ATP or x 2= 6

• - Oxidation of glyceraldehyde phospate to 1,3 – bisphosphoglycerate

• - 3-Bisphosphoglycerate is an acyl phosphate and is a high energy compound.

• aldehyde group.of glyceraldehyde 3-phosphate is dehydrogenated to a carboxylic anhydride with phosphoric acid

- reaction is similar to hemiacetal formation but product is thiohemiacetal

- NADH formed is reoxidized to NAD

STEP 71,3-bisphosphoglycerate + ADP ↔ 3- phosphoglycerate + ATP

PHOSPHOGLYCERATE KINASE catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP through the process of PHOSPHORYL TRANSFER

Formation of ATP is referred to as substrate-level phosphorylation because the phosphate donor, 1,3-BPG is a substrate with high phosphoryl-transfer potential.

STEP 8

3-phosphoglycerate ↔ 2-phosphoglycerate

the enzyme used in the reaction is a MUTASE alters the confuguration by changing the position

of the phosphoryl group phosphoenolpyruvate is formed by dehydration of

2-phosphoglycerate

• Reactions 7 and 8

– Phosphoglycerate kinase assists in the hydrolysis of 1,3-bisphosphoglycerate, and uses the energy to form one ATP (2 to double).

– Transfers phosphate from 3 BPG to ADP(substrate level phosphorylation)

- arsenic- competes with inorganic PO4 in the reaction

– Phosphoglucomutase moves the

phosphate from the 3 position to the 2

position.

STEP 9

2-phosphoglycerate ↔ phosphoenolpyruvate + H2O

ENOLASE catalyzes the formation of phosphoenolpyruvate (PEP)

elevates the transfer potential of the phosphoryl group

STEP 10 (IRREVERSIBLE)

Phosphoenolpyruvate + ADP + H+ pyruvate + ATP

• Reactions 9 and 10

– Enolase splits off a water and forms a second high energy phosphate called phosphoenolpyruvate (PEP).

– Inhibited by fluoride

– The energy from PEP is used to phosphorylate ADP to form ATP. The enzyme involved is pyruvate kinase, and the hydrolysis product is pyruvate

• - phosphate from phosphoenolpyruvate is transferred to ADP (substrate level phosphorylation)

PYRUVATE KINASE is the enzyme used in this reaction.

Table 2: ATP Production Table 3: Net Production of ATP

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1. Glucose produces two pyruvate moleculesbecause of Step 4, glucose (6 carbon molecule) was cleaved into TWO 3-carbon molecules2. ATP is initially requiredATP was used in Steps 1 and 33. ATP is producedNADH was produced in Step 6 ATP was produced in Steps 7 & 104. Fate of NADH+ Enters the Electron Transport Chain to produce 3 ATP

Note:steps 6, 7, and 10 were multiplied by two to account for the two 3 carbon sugars formed in Step 4

Pathway of Glycolysis:

Glucose

ATPADPHexokinase / Glucokinase

Glucose 6-Phosphate

Phosphohexose Isomerase

Fructose 6-Phosphate

ATPADPPhosphofructokinase (PFK1)

Fructose-1,6-Biphosphate

Aldolase

DHAP GLYC-3-P Phosphotriose Isomerase

Phosphoric AcidOXIDATION STEP NAD

NADH (lost and used later)Glyceraldehyde-3-Phosphate Dehydrogenase

1,3-BPG

ADP SUBSTRATE LEVEL ATP (ATP is formed)PHOSPHORYLATION Phosphoglycerate Kinase

3 Phosphoglycerate ISOMERIZATION Phosphoglycerate Mutase

(transfer of Phosphate)

2 Phosphoglycerate

DEHYDRATION H2OEnolase

Phosphoenolpyruvate

SUBSTRATE LEVEL ADP + Pi PHOSPHRYLATION ATP

Pyruvate kinase

PYRUVATE

REDUCTION Lactate DehydrogenaseNADH (regenerated)NAD

LACTATE

Summary of glycolytic pathway:

1. Glucose is Phosphorylated to Glucose 6-P - uses ATP and liberates ADP

2. Glucose-6-Phosphate is converted to Fructose-6-phosphate -Enzyme: Phosphohexose Isomerase -involves an aldose-ketose isomerization

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STEP ATP (used -; produced +)1 -13 -16-NADH +3 * 2 = 67 +1 * 2 = 210 +1 * 2 = 2Total 8 ATP

3. Fructose-6-Phosphate is converted to Fructose-1,6-Biphosphate -phosphorylation with ATP catalyzed by Phosphofructokinase (PFK-1) -functionally irreversible and inducible (subject to allosteric regulation) -major role in regulating the rate of glycolysis

4. Fructose 1,6-Biphosphate cleaved into: Glyceraldehyde-3-phosphate and dihydroxyacetone Phosphate -cleaved by Aldolase (fructose 1,6-biphosphate aldolase)

5. DHAP and GLYC-3P are inter-converted by the enzyme Phosphotriose Isomerase

6. Glyceraldehyde-3-Phosphate Oxidized to 1,3-Biphosphoglycerate

a. Glyceraldehyde is first Oxidized into an Acid (intermediate)

b. Acid is then converted into 1,3 Bisphosphoglycerate (High Energy Compound)

-Enzyme: Glyceraldehyde-3-Phosphate Dehydrogenase

NAD-dependent Consists of 4 identical polypeptides (monomers)

forming a tetramer with -SH groups present on each polypeptide

one of the –SH groups is the active site of the enzyme

combines with the substrate forming a thiolhemiacetal that is oxidized to a thiol ester

-Hydrogens removed in this oxidation are transferred to NAD+-Pi is added forming 1,3-biphosphoglycerate, and the –SH group is reconstituted

7. 1,3-Biphosphoglycerate forms 3-Phosphoglycerate - phosphate transferred from 1,3-biphosphoglycerate onto ADP, forming ATP (substrate-level phosphorylation) and 3-phoshoglycerate - Enzyme: Phosphoglycerate Kinase - 1,3-BPG is a High Energy Compound (with ADP + Kinase, ATP can be formed - 2 molecules of triose phosphate are formed per molecule of glucose, 2 molecules of ATP are generated at this stage per molecule of glucose undergoing glycolysis - Toxicity of Arsenic is due to competition of arsenate with inorganic phosphate (Pi) in the above reactions to give 1-arseno-3-phosphoglycerate, which hydrolyzes spontaneously to give 3- phosphoglycerate plus heat, without generating ATP

**Biphosphoglycerate Mutase-catalyzes the conversion of 1,3-biphosphoglycerate to 2,3-Biphosphoglycerate which is converted to 3-phosphoglycerate by Phosphoglycerate Mutase (next step) - this alternative pathway serve to provide 2,3-biphosphoglycerate

**2,3-Biphosphoglycerate (BPG) -binds to hemoglobin, decreasing its affinity for oxygen

8. 3-Phosphoglycerate is Isomerized to 2-Phosphoglycerate -Enzyme: Phosphoglycerate Mutase -2,3-biphosphoglycerate is an intermediate of this reaction (2,3 BPG)

9. 2-Phosphoglycerate forms Phosphoenolpyruvate -dehydration of 2-phosphoglycerate forming phosphoenolpyruvate (loss of H2O) -Enzyme: Enolase -inhibited by fluoride

10. Phosphoenolpyruvate Forms Pyruvate Acid

-phosphate of phosphoenolpyruvate transferred to ADP -Enzyme: Pyruvate kinase -irreversible in physiological conditions -product of enzyme-catalyzed reaction enolpyruvate, undergoes spontaneous isomerization to pyruvate not available to undergo the reverse reaction

11. Pyruvate forms Lactic Acid -Enzyme: Lactate Dehydrogenase -Pyruvate uses NADH lost earlier to form Lactic Acid

REGULATION OF GLYCOLYSIS

-Glycolysis is regulated at Three Steps Involving Nonequilibrium Reactions

-most of the reactions of glycolysis are reversible

-3 are markedly Exergonic; Considered Irreversible:o Hexokinase and Glucokinase-

inhibited by its own product, G-6-P, in high levels

o Phosphofructokinase (PFK1)- stimulated by high levels of AMP or by insulin, inhibited by high levels of ATP or citrate; inhibition relieved by 5’AMP

o Pyruvate kinase- inhibited by high levels of ATP

- In erythrocytes, reaction catalyzed by phosphoglycerate kinase may be bypassed by reaction of bisphosphoglycerate mutase (conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate) and 2,3-bisphosphoglycerate phosphatase (hydrolysis to 3-phosphoglycerate and Pi). There is therefore no net yield of ATP.

Oxidation of Pyruvate to Acetyl-CoA is the irreversible route from Aerobic Glycolysis to the citric acid cycle

Pyruvate formed in cytosol, transported into mitochondrion by a proton symporter

Inside mitochondrion, pyruvate is oxidatively decarboxylated to acetyl-CoA by multienzyme complex, associated with the inner mitochondrial membrane

Pyruvate Dehydrogenase Complex is analogous to Alpha-Ketoglutarate Dehydrogenase complex of citric acid cycle

Pyruvate is decarboxylated by Pyruvate Dehydrogenase to a Hydroxyethyl Derivative to thiazole ring of enzyme-bound Thiamin Diphosphate

Hydroxyethyl reacts with Oxidized Lipoamide (prosthetic group of dihydrolipoyl transacetylase) to form Acetyl Lipoamide

Acetyl lipoamide reacts with Coenzyme A to form Acetyl-CoA and Reduced Lipoamide

Cycle of reaction is completed when the reduced lipoamide is reoxidized by a flavoprotein: dihydrolipoyl dehydrogenase, containing FAD

Reduced flavoprotein is oxidized by NAD+, transfers reducing equivalents to the respiratory chain:

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Pyruvate + NAD+ + CoA Acetyl-CoA + NADH + H+ +CO2

**Pyruvate Dehydrogenase Complex -consists of a number of polypeptide chains of each of the three component enzymes, all organized in a regular spatial configuration -inhibited by its products acetyl-CoA and NADH

Figure 1: 2,3-Bisphosphoglycerate pathway in erythrocytes (Image taken from: http://web.indstate.edu/thcme/mwking/hemoglobin-myoglobin.html).

GLYCOLYSIS TO TCAC irreversible route – oxidation of pyruvate to acetyl-

CoA pyruvate from the cytosol is transported into the

mitochondrion and is oxidatively decarboxylated by a pyruvate dehydrogenase complex

pyruvate dehydrogenase is regulated by:o inhibition by its products, acetyl-CoA and NADHo phosphorylation by a kinase of three serine

residues, resulting in decreased activityo dephosphorylation by a phosphatase, causing

increased activity

CLINICAL ASPECTS

inhibition of pyruvate dehydrogenase may result to lactic acidosis. This may be caused by any of the following:

- Arsenite and mercuric ions react with the –SH group of lipoic acid and inhibit pyruvate dehydrogenase, as does a dietary deficiency of thiamin, allowing pyruvate to accumulate

- nutritionally deprived alcoholics are thiamin-deficient, may develop potentially fatal pyruvic and lactic acidosis

- inherited pyruvate dehydrogenase deficiency due to defects in one or more of the components of the enzyme complex, present with lactic acidosis after a glucose load

- because of its dependence on glucose as a fuel, the brain is a prominent tissue where these metabolic defects manifest themselves in neurologic disturbances

- inherited aldose A deficiency and pyruvate kinase deficiency in erythrocyte cause hemolytic anemia

- exercise capacity of patients with muscle phosphofructokinase deficiency is low especially in low carbohydrate diets

- by providing an alternative lipid fuel when blood free fatty acids and ketone bodies are increase, work capacity is improved

Hemolytic Anemias – diseases in which enzymes of glycolysis are deficient

Fatigue – if defect affects skeletal muscle Lactate is used for gluconeogenesis in the

liver, an energy-expensive process responsible for much of the hypermetabolism seen in cancer cachexia

Lactic Acidosis – results from impaired activity of pyruvate dehydrogenase

Table 4: Summary

Step Reaction Enzyme Reaction Type1 Glucose + ATP glucose 6-phophate + ADP + H+ Hexokinase/Glucokinase Phosphoryl transfer2 Glucose 6-phosphate ↔ fructose 6-phophate + ADP +H+ Isomerase Isomerization3 Fructose 6-phosphate + ATP fructose 1,6 bisphophate

+ ADP + H+Phosphofructokinase Phosphoryl transfer

4 Fructose 1,6-bisphosphate ↔ dihydroxyacetone phosphate + glyceraldehyde 3-phosphate

Aldolase Aldol cleavage

5 Dihydroxyacetone phosphate ↔ glyceraldehydes 3-phosphate

Triose phosphate isomerase

Isomerization

6 Glyceraldehyde 3-phosphate + Pi + NAD ↔ 1,3-bisphosphoglycerate + NADH+ + H+

Glyceraldehyde 3-phopsphate dehydrogenase

Phosphorylation coupled to oxidation

7 1,3-bisphosphoglycerate + ADP ↔ 3-phosphoglycerate + ATP

Phosphoglycerate kinase Phosphoryl transfer

8 3-phosphoglycerate ↔ 2-phosphoglycerate Mutase Phosphoryl shift9 2-phosphoglycerate ↔ phosphoenolpyruvate + H2O Enolase Dehydration10 Phosphoenolpyruvate + ADP + H+ pyruvate + ATP

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