Principles of Energy Harvest

• Endothermic

–Photosynthesis

• Exothermic

–Glycolysis

–Respiration

6CO2 + 6H2O

2840 kJ EnergyAs Heat

Combustion of glucose in a bomb calorimeter

Glucose

Aerobic Respiration

• Products would be the same if the glucose were burned in a bomb calorimeter with oxygen

• A redox reaction• Glucose is oxidized• Oxygen is reduced• Cells carry out a redox process in about 30

steps• Electrons associated with hydrogen atoms in

glucose are transferred to oxygen

Redox reactions

• Oxidation-reduction• OIL RIG

(adding e- reduces + charge)

• Oxidation is e- loss; reduction is e- gain

• Reducing agent:e- donor

• Oxidizing agent:e- acceptor

Redox Reactions

• Reaction in which one substance transfers one or more electrons to another substance is called oxidation-reduction reaction– Redox for short

• Gain or 1 or more electrons by atom, ion, or molecule is called reduction

• Loss of 1 or more electrons by atom, ion, or molecule is called oxidation

Cellular respiration

• Glycolysis: cytosol; degrades glucose into pyruvate

• Kreb’s Cycle: mitochondrial matrix; pyruvate into carbon dioxide

• Electron Transport Chain: inner membrane of mitochondrion; electrons passed to oxygen

Overview of Glycolysis

The Embden-Meyerhof (Warburg) Pathway • Discovered by Hans Buchner and Eduard Buchner when

sucrose was found rapidly fermented into alcohol by yeast;• Essentially all cells carry out glycolysis• Enzyme driven• Site of glycolysis is in cytosol; • Ten reactions - same in all cells - but rates differ • Two phases:

– First phase converts glucose to two G-3-P – Second phase produces two pyruvates

• Products are pyruvate, ATP and NADH • Three possible fates for pyruvate

Glycolysis

• 1 Glucose 2 pyruvate molecules

• Energy investment phase: cell uses ATP to phosphorylate fuel

• Energy payoff phase: ATP is produced by substrate-level phosphorylation and NAD+ is reduced to NADH by food oxidation

• Net energy yield per glucose molecule: 2 ATP plus 2 NADH; no CO2 is released; occurs aerobically or anaerobically

Kreb’s Cycle• If molecular oxygen is present…….• Each pyruvate is converted into acetyl

CoA (begin w/ 2): CO2 is released; NAD+ ---> NADH;

coenzyme A (from B vitamin), makes molecule very reactive

• From this point, each turn 2 C atoms enter (pyruvate) and 2 exit (carbon dioxide)

• Oxaloacetate is regenerated (the “cycle”)

• For each pyruvate that enters: 3 NAD+ reduced to NADH; 1 FAD+ reduced to FADH2 (riboflavin, B vitamin); 1 ATP molecule

Electron transport chain

• Cytochromes carry electron carrier molecules (NADH & FADH2) down to oxygen

• Chemiosmosis: energy coupling mechanism

• ATP synthase: produces ATP by using the H+ gradient (proton-motive force) pumped into the inner membrane space from the electron transport chain; this enzyme harnesses the flow of H+ back into the matrix to phosphorylate ADP to ATP (oxidative phosphorylation)

Review: Cellular Respiration

• Glycolysis: 2 ATP (substrate-level phosphorylation)

• Kreb’s Cycle: 2 ATP (substrate-level phosphorylation)

• Electron transport & oxidative phosphorylation: 2 NADH (glycolysis) = 6ATP 2 NADH (acetyl CoA) = 6ATP 6 NADH (Kreb’s) = 18 ATP

2 FADH2 (Kreb’s) = 4 ATP

• 38 TOTAL ATP/glucose

4 stages of aerobic respiration

Stage 1: Glycolysis

Stage 2: Formation of Acetyl coenzyme A

Stage 3: The Citric Acid Cycle

Stage 4: Electron transport chain

A Road Map for Cellular RespirationCytosol

Mitochondrion

High-energyelectronscarriedby NADH

High-energyelectrons carriedmainly byNADH

Glycolysis

Glucose2

Pyruvicacid

KrebsCycle

ElectronTransport

Glycolysis and Fermentation

Glycolysis: Embden Meyerhof pathway

• The pathway for lactate fermentation in muscle is the same pathway as alcohol fermentation, showing an underlying unity in biology.

• Gustav Embden

• Otto Meyerhof

• Jacob Parness

1922 Nobel Prize

Related metabolic processes

Fate of Pyruvate

2 Pyruvic acid

Overview of Glycolysis

GLYCOLYSIS GLYCOLYSIS Glucose ATP hexokinase ADP Glucose 6-phosphate phosphogluco- isomerase Fructose 6-phosphate ATP phosphofructokinase ADP Fructose 1,6-bisphosphate aldolase

triose phosphate isomerase Dihydroxyacetone Glyceraldehyde phosphate 3-phosphate

Glyceraldehyde 3-phosphate glyceraldehyde NAD+ + Pi

3-phosphate NADH + H+

dehydrogenase 1,3-Bisphosphoglycerate  ADP phosphoglycerate kinase ATP 3-Phosphoglycerate phosphoglyceromutase 2-Phosphoglycerate enolase H2O Phosphoenolpyruvate ADP pyruvate kinase ATP Pyruvate

Glycolysis: 1

Glycolysis: stage 1

The three steps ofstage 1 begin withthe phosphorylationof glucose byhexokinase

Energy used,none extracted

ATP ADP

glucose

glucose 6-phosphate

∆Go = -16.7 kJ/mole

Step 1: Adding a phosphate

Enzyme:

hexokinase

Phosphoryl transfer reaction. Kinases transfer phosphate from ATP to an acceptor. Hexokinase has a more general specificity in that it can transfer phosphate to other sugars such as mannose.

ΔG°’= -4.0 kcal mol-1

Glucose phosphorylation: step 1

Glucose is a relatively stable molecule and is not easily broken down. The phosphoylated sugar is less stable.

ATP serves as both source of phosphate and energy needed to add phosphate group to the molecule.

Induced fit in hexokinase

Conformation Changes on binding glucose,

the two lobes of theenzyme come together and Surround the substrate

Step 2: Isomerization

glucose 6-phosphate fructose

6-phophate

aldose to ketose isomerization reversible, G°´= 1.7 kJ/mole

6 carbon ring

5 carbon ring

Enzyme: phosphoglucoisomerase

The conversion of an aldose to a ketose.

Phosphoglucose Isomerase

ΔG°’= .40 kcal mol-1

Formation of fructose-6-phosphate: step 2

by phosphoglucose isomerase

The enzyme opens the ring, catalyzes the isomerization, and promotes the closure of the five member ring.

Step 3: Second phosphorylation

-second ATP investment-highly exergonic, essentially irreversible, G°´= -14.2 kJ/mole

fructose 1,6 bisphosphate

ATP ADP

fructose 6-phosphate

Enzyme: phosphofructokinase

Phosphofructokinase-1 PFK

ΔG°’= -3.4 kcal mol-1

The 2nd investment of an ATP in glycolysis.

Bis means two phosphate groups on two different carbon atoms. Di means two phosphate groups linked together on the same carbon atom.

PFK is an important allosteric enzyme regulating the rate of glucose catabolism and plays a role in integrating metabolism.

Formation of fructose 1,6-bisphosphate: step 3

by phosphofructokinase (PFK): an allosteric enzyme that regulates

the pace of glycolysis.

Allosteric Enzymes• Part II. Allosteric Regulation • Control of Enzyme Activity by Non-Covalent Modifiers is usually called

allosteric regulation since the modifier binds to the enzyme at a site other than the active site but alters the shape of the active site. Allosteric is a word derived from two Greek words: 'allo' meaning other and 'steric' meaning place or site; so allosteric means other site and an 'allosteric enzyme' is one with two binding sites - one for the substrate and one for the allosteric modifier molecule, which is not changed by the enzyme so it is not a substrate. The molecule binding at the allosteric site is not called an inhibitor because it does not necessarily have to cause inhibition - so they are called modifiers. A negative allosteric modifier will cause the enzyme to have less activity, while a positive allosteric modifier will cause the enzyme to be more active. In order for allosteric regulation to work, the enzyme must be multimeric (ie. a dimer, trimer, tetramer etc.). The concept is easily illustrated using a dimer as the model system, but it applies equally well to higher order multimers such as trimers and tetramers, etc.

Glycolysis: stage 2

Two 3-carbon fragments are produced from one6-carbon sugar

No energyused orextracted

Step 4: Cleavage to two triose phosphates

Reverse aldol condensation; converts a 6 carbon atom sugar to 2 molecules, each containing 3 carbon atoms.

Enzyme: aldolasealdolase

Aldol Condensation• An Aldol condensation is an organic reaction in which an

enolate ion reacts with a carbonyl compound to form a β-hydroxyaldehyde or β-hydroxyketone, followed by dehydration to give a conjugated enone.

•

• Aldol condensations are important in organic synthesis, providing a good way to form carbon–carbon bonds. The Robinson annulation reaction sequence features an aldol condensation; the Wieland-Miescher ketone product is an important starting material for many organic syntheses. Aldol condensations are also commonly discussed in university level organic chemistry classes as a good bond-forming reaction that demonstrates important reaction mechanisms. In its usual form, it involves the nucleophilic addition of a ketone enolate to an aldehyde to form a β-hydroxy ketone, or "aldol" (aldehyde + alcohol), a structural unit found in many naturally occurring molecules and pharmaceuticals.

• Enol: An organic compound containing a hydroxyl group bonded to a carbon atom, which in turn is doubly bonded to another carbon atom.

Cleavage of six-carbon sugar: step 4

Glycolysis: 2

Step 5: Isomerization of dihydroxyacetone phosphate

H2C-OH C=O

CH2-O- P

dihydroxyacetone glyceraldehyde phosphate 3-phosphate

Enzyme: triose-phosphate isomerase

Salvage of three-carbon fragment: step 5

Glycolysis: stage 3

The oxidation ofthree-carbon fragmentsyields ATP

Energyextracted,2x2 ATP

Glycolysis: 3

Step 6: Formation of 1,3-Bisphosphoglycerate

Done in two steps

glyceraldehyde 3-phosphate 1,3 bisphosphoglycerate

Enzyme: glyceraldehyde-3-phosphate dehydrogenase

addition of phosphate, oxidation, production of NADH, formation of high energy compound

The fate of glyceraldehyde 3-phosphate

Stage 3: The energy yielding phase.

Glyceraldehyde 3-phosphate DH

ΔG°’ = 1.5 kcal mol-1

1,3-BPG has a high phosphoryl-transfer potential. It is a mixed anhydride.

An aldehyde is oxidized to carboxylic acid and inorganic phosphate is transferred to form acyl-phosphate. NAD+ is reduced to NADH.

Notice, under anaerobic conditions NAD+ must be re-supplied.

Glyceraldehyde 3-phosphate dehydrogenase

Active site configuration

Step 7: Transfer of phosphate to make ATP Formation of ATP from 1,3-Bisphosphoglycerate:

Enzyme: phosphoglycerate kinase

first substrate level phosphorylation, yielding ATP2 1,3 bis PG yield 2 ATPs, thus ATP yield = ATP inputhigh free energy yield, G°´= -18.8kJ/mole drives several of the previous steps

7: Phosphoglycerate KinaseSubstrate-level phosphorylation

ΔG°’ = -4.5 kcal mol-1ATP is produced from Pi and ADP at the expense of carbon oxidation from the glyceraldehyde 3-phosphate DH reaction.

Remember: 2 molecules of ATP are produced per glucose.

At this point 2ATPs were invested and 2ATPs are produced.

Two-process reaction

Aldehyde Acid

Step 8: Phosphate shift setup

- shifts phosphate from position 3 to 2- reversible, ΔG = + 4.6 kJ/mole

Enzyme:

phosphoglycerate mutase

Phosphate shift

Phosphoglycerate mutase

ΔG°’ = 1.1 kcal mol-1

Step 8: Rearrangement

Step 9: Removal of Water leadsto formation of double bond

little energy change in this reaction, ΔG = + 1.7 kJ/mole because the energy is locked into enolphosphate. Phosphate group attached by unstable bond, therefore high energy

Enzyme:

enolase

Generation of second very high energy compound by a dehydration reaction

Enolase

ΔG°’ = .4 kcal mol-1

Dehydration reactionPEP

the energy is locked into the high energy unfavorable enol configuration by phosphoric acid ester

An enol phosphate is formed: step 9

Dehydration elevates the transfer potential of the phosphoryl group, which traps the molecule in anunstable enol form

Enol: molecule with hydroxyl group next to double bond

Step 10: Formation of Pyruvate & ATP

Enzyme: pyruvate kinase

phosphoenolpyruvate pyruvate

second substrate level phosphorylation yielding ATPhighly exergonic reaction, irreversible, ΔG = -31.4 kJ/mole.

•Substrate level phosphorylation is the synthesis of ATP from ADP that is not linked to the electron transport system.

Pyruvate Kinase2nd example of substrate level phosphorylation.

The net yield from glycolysis is 2 ATP

ΔG°’ = -7.5 kcal mol-1

unstable Enol form more stable keto form

PEP

Maintaining Redox Balance

NAD+ must be regeneratedfor glycolysis to proceed

Glycolysis is similar in all cells,the fate of pyruvate is variable

Diverse fates of pyruvate

To citric acid cycle

The Conversion of Glucose to PyruvateGlucose + 2 Pi + 2 ADP + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH +2 H+

The Energy released from the anaerobic conversion of glucose to pyruvate is

-47kcal mol-1.

Under aerobic conditions much more chemical bond energy can be extracted from

pyruvate.

The question still remains: How is NAD+ supplied under anaerobic conditions? Or how

is redox balance maintained?

Under anaerobic conditions pyruvate is converted to lactate. Exercising muscle is an example.

The NAD+ that is consumed in the glyceraldehyde 3-phosphate reaction is produced in the lactate DH reaction. The redox balance is maintained. The activities of glyceraldehyde 3-phosphate DH and Lactate DH are linked metabolically.

What happens to the lactate after a run?

ATP

In anaerobic yeast, pyruvate→ethanol

Pyruvate is decarboxylated.

Acetaldehyde is reduced.

Variations on a theme in alcoholic fermentation.

Here also, there is no net oxidation reduction.

Enzyme ClassificationDehydrogenase- oxidizes substrate using cofactors aselectron acceptor or donor (pyruvate dehydrogenase)Reductase- adds electrons from some reduced cofactor(enoyl ACP reductase)Kinase- phosphorylates substrate (hexokinase) Hydrolases - uses water to cleave a molecule Phosphatase- hydrolyzes phosphate esters (glucose-6-phosphatase) Esterase (lipase)- hydrolyzes esters (those that act on lipid esters are lipases) (lipoprotein lipase) Thioesterases - hydrolyzes thioestersThiolase- uses thiol to assist in forming thioester (β-ketothiolase)Isomerases- interconversions of isomers

2

Pyruvicacid

Aceticacid

Coenzyme A

Acetyl-CoA(acetyl-coenzyme A)

CO2

Stage 2: The Krebs Cycle

• The Krebs cycle completes the breakdown of sugar• In the Krebs cycle, pyruvic acid from glycolysis is first

“prepped” into a usable form, Acetyl-CoA

Acetyl-CoA(acetyl-coenzyme A)

CO2

pyruvic acidacetic acid

Figure 6.11

Input

3 NAD

FAD

KrebsCycle

Output

2 CO2

1 2

3

4

5

6

input output

ADP

NAD+

FAD

Figure 6.12

Proteincomplex

Electroncarrier

Innermitochondrialmembrane

Electronflow

Electron transport chain ATP synthase

Electron Transport Chain

Stage 3: Electron Transport

• Electron transport releases the energy your cells need to make the most of their ATP

• The molecules of electron transport chains are built into the inner membranes of mitochondria– The chain functions as a chemical machine that uses

energy released by the “fall” of electrons to pump hydrogen ions across the inner mitochondrial membrane

– These ions store potential energy

Adding Up the ATP from Cellular Respiration

Figure 6.14

Cytosol

Mitochondrion

Glycolysis

Glucose2

Pyruvicacid

2Acetyl-

CoA

KrebsCycle

ElectronTransport

bydirectsynthesis

by directsynthesis

byATPsynthase

Maximumper

glucose:

Figure 6.13

Food

Polysaccharides Fats Proteins

Sugars Glycerol Fatty acids Amino acids

Amino groups

Glycolysis Acetyl-CoA

KrebsCycle Electron

Transport

ATP hydrolysis and synthesis