Introduction Free Energy Bioenergetics

BIOC13: Bioenergetics & Metabolism

• Prof. Michelle Aarts

– maarts@utsc.utoronto.ca

• Office Hours: Tuesdays 2 – 4pm in SW525

• Contact me by e‐mail:

– for appointments outside office hours

– for problems, conflicts or missed tests & assignments

Recommended Text

• Fundamentals of Biochemistry: Life at the Molecular Level, 3rd Edition

• by Voet, Voet, and Pratt

• Wiley Publishing

• Online text and resources available

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Texts available used (equivalent info)

• Biochemistry 5th or 6th ed.– Berg, Stryer, Tymoczko

• Principles of Biochemistry, 3rd or 4th ed.

–Horton, Moran, Scrimgeour, Perry & Rawn

• Lehninger – Principles of Biochemistry

– Nelson and Cox

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Other Texts and Resources

• Biochemistry– Voet & Voet (Wiley)

• Fundamentals of Biochemistry 2nd– Voet, Voet & Pratt (Wiley)

• Biochemistry: The Molecular Basis of Life – McKee & McKee

• Publisher websites– Pearson-Prentice Hall– Wiley– whfreeman

• Websites (see course intranet site)

• Additional information, animations, 3D images– IUBMB (metabolic maps and

animations by Donald Nicholson)www.iubmb.org

– EXPasy (protein database)www.expasy.org

– KEGG pathway maps www.genome.jp/kegg/

– www.enzyme-database.org

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Posted on the Intranet

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Syllabus & Course information

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Take Home Questions•

Can you answer these questions for each “Topic”

that 

we cover in class?

•

Lecture topics (1 – 9)

•

Supplemental reading

•

Web resources

•

Problem sets & answers

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Evaluations!• Quizzes (4) 

– Jan 26th, Feb 16th, March 16th, April 6th

– together 20% of final grade– 5 –10 questions (MC and written)

• Midterm– 30% of final grade– MC, fill‐in‐the‐blanks, short answer

• Final Exam– 50% of final grade (cumulative content)– MC, fill‐in‐the‐blanks, short answer

• Please refer to Syllabus for dates and content

BGYC13H Prerequisite Check

**If you are lacking a pre-requisite you must come see me in person**

Pre-requisites– BGYB10, BGYB11– CHMB41, CHMB42 (organic chemistry)

BGYC13 & CHMB42 are required courses for:– Specialist & Co-op in Cell & Molecular Biology– Specialist & Co-op in Neuroscience– Specialist in Biological Chemistry– Major in Biochemistry– **Specialist in Human Biology (BGYC12 or C13)

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Metabolism and Bioenergetics

• This week:– Overview of metabolism (Ch. 14)– Overview of key reaction pathways and systems– Concept of bioenergetics and energy currency (ATP)

(Chapters 1 & 14)– Redox reactions (Ch. 14)

– Overview of course topics

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Metabolism & Bioenergetics

• Metabolism– The total network of chemical reactions carried out by a living

cell• Anabolic (biosynthesis)• Catabolic (degradation)• Metabolites

• Bioenergetics– Biochemical transformation of energy

• Usually metabolism of ATP (NADH, NADPH)• Most important pathways are membrane-associated electron

transport in oxidative phosphorylation and photosynthesis

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Anabolic Reactions Catabolic Reactions

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Metabolic Types

• Autotrophs

– (H2O, CO2, NH3, H2S)– Photoautotroph (plants)– Chemoautotroph (bacteria) – (NH3 , H2S, Fe2+)

• Heterotrophs

– Photoheterotroph (some bacteria) – Chemoheterotroph (animals)

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Why Study Metabolism?

• It’s Important!– “A bewildering array of biochemical reactions occur in any living

cell yet the principles that govern metabolism are the same in all organisms; a result of their common evolutionary origin and the constraints of the laws of thermodynamics. In fact, many of the specific metabolic reactions are common to all organisms, with variations due primarily to differences in the source of free energy that supports them.”

– Voet, Voet & Pratt, 2006

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Why Study Metabolism?

• Enzymes account for a significant proportion of expressed genes in any organism– E. coli

• 4400 genes, 1560 enzymes (35%), 900 intermediary metabolic enzymes (21%)

– C. elegans• 19,100 genes, 5300 metabolic enzymes (28%)

– Drosophila• 14,100 genes, 2400 metabolic enzymes (17%)

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Why Study Metabolism? (a.k.a. what you should get out of this course)

• What’s involved?– Thermodynamics, free energy– Reaction kinetics, pathway flux, regulatory

mechanisms– Enzymes (what do they do? How do they

work?)– Biosynthesis and breakdown of organic

compounds– Oxidation and reduction reactions– Electron Transport and Photosynthesis

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What are Metabolic Pathways?

• “A series of chemical reactions where the product of one reaction becomes the substrate for the next reaction.”

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Types of Metabolic Pathways

Linear Cyclic Spiral

* Most pathways have branch points - isolated, linear pathways are rare

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Donald Nicholson, University of Leeds

© IUBMB 200317

The “hub” of metabolism – D. Nicholson (IUBMB) 2002

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Metabolic Pathways – Key Features?

– Individual reactions must be specific (i.e. one product)– An entire series of reactions must be

thermodynamically favorable– Pathways proceed in one direction (irreversible)

• Anabolic and Catabolic pathways differ• Pathways contain a committed step

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Metabolic Pathways = Why?

CO2 + H2O ↔ glucose

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Metabolic Regulation – Why?

• Control the Flux of metabolites through a pathway– Allows response to:

• Changes in environment• Supply of energy and nutrients• Genetic programming

• Most metabolic pathways occur in a single direction under physiologic conditions (irreversible) – thus are far from thermodynamic equilibrium

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A B C D PE1 E2 E3 E4

4. Enzyme expression(genetic/degradation)

Regulation of metabolic pathways occurs at the level of rate limiting enzymes

2. Covalent modification1. Allosteric Control

3. Substrate cycles

How are Metabolic Pathways Regulated?

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Metabolic Pathway Regulation by compartmental separation

• Concentrate enzymes & cofactors

• Movement of metabolites between compartments

• Co-ordinate enzyme regulation locally

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Bioenergetics

• Study of the changes in energy during metabolic reactions

• Organisms need an input of free energy for:– Mechanical work– Active transport of molecules– Biosynthesis

• Metabolism is essentially composed of ‘coupled’, interconnecting reactions

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Bioenergetic Systems

• Biochemical reactions• Photosynthesis• Oxidative Phosphorylation (Electron Transport

Chain)• ATP synthesis

– Thermodynamics (free energy, ∆G)

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Laws of Thermodynamics govern biochemical systems

• “Total energy in a system and its surroundings is constant”

• “potential energy” – likelihood that 2 atoms will react with one another

• “Total entropy (disorder) of a system and its surroundings always increases”

• Entropy (S) of a system can decrease if the S of the surroundings increases

• Decreased S is accomplished by the release of heat (H, enthalpy)

∆H↓ ∆Ssystem →↑ ∆Ssurroundings

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Gibbs Free Energy Change (∆G)

• A measure of the energy available from a reaction

• Standard Gibbs Free Energy Change (∆Gº)

• Actual Gibbs Free Energy change (∆G)

∆G = ∆H - T∆S

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Free Energy Changes (∆G)

• Reactions will occur spontaneously in vivo when ∆G<0 (negative) (take home message from thermodynamics)

• When ∆G is positive the reaction requires energy input to proceed in the direction written

A + B → C + D

• Reactions at equilibrium (no net change) have a ∆G approaching zero

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Gibbs Free Energy

•

Derivation of the equations and how to use them  next class

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• The Standard Gibbs free energy difference (∆Gº) tells us if a reaction in one direction is favorable when the concentrations of both the substrates and products is 1.0M.

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However…• The difference between ∆G and ∆Gº depends on the cellular

conditions – most importantly concentrations

• The actual Gibbs free energy difference (∆G) tells us if the reaction is favorable when the [substrates] and [products] are something other than 1.0M (Q = mass action ratio).

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Free energy at equilibrium

• Concentrations at equilibrium (Keq) brings the free energy difference (∆G) between substrates and products to zero, so there is no net production in either direction.

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• “When flux through a pathway changes, the intracellular concentrations of metabolites vary.”– Physiologic changes are relatively small– Most enzymes catalyze near-equilibrium reactions– Can restore balance quickly

∆Gº’ positive

∆Gº’ ~ 0

∆Gº’ negative

Metabolic Flux and Equilibrium

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• Near equilibrium reactions are influenced by changes in the [substrates] and [products] without changing flux through the pathway– Not a good control point

• [substrates] and [products] have little effect on flux through irreversible reactions

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Role of Enzymes in Metabolic reactions

• Couple reactions• Stabilize Transition states (decrease energy)

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• Enzymes stabilize high energy transition states that would make a reaction unfavorable

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Enzymes have common features

• High substrate specificity• Active site

• 3-D cleft that binds substrate• Small portion of total protein• Multiple intermolecular sites of attraction with substrate• Unique microenvironment

• Conformational change on binding• Regulatory sites (specific inhibition)• Use of cofactors or co-enzymes

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Enzymes and reactions recur throughout metabolism

• Coupled reactions• Activated carriers• Recurring use of enzyme families and reaction types

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Enzyme Classifications

Enzyme Class Reaction Catalysed

Oxidoreductases Oxidation-reduction

Transferases Move functional groups

Hydrolases Hydrolysis

Lyases Eliminate group & form double bond

Isomerases Isomerization

Ligases Bond formation (ATP coupled)

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Some enzymes need cofactors• Amino acid side chains may 

participate in reaction

• Small molecules (Mg2+, Zn2+) stabilize substrate binding

• Coenzymes:

– May be added to substrate (coenzyme A)

– Soluble (ATP, NAD+)

– Bound prosthetic groups (FAD, FMN, biotin, 

Coenzymes and Cofactors can be derived from Vitamins

• Generally “B”Vitamins

Enzymes require cofactors (many derived from vitamins)

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Activated carriers or Co-factors

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Coenzyme Acarboxybiotin

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Metabolic Pathways -

Energetic Coupling

•

A thermodynamically unfavorable reaction (+ΔG or  Keq

that favors substrates) can be driven forward by  coupling to a favorable reaction

•

1 

•

2 

•

3

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Serial Coupling

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Coupling 2 Reactions

•

2 individual reactions occur in the same enzyme  active site (2 substrates, 2 products)

–

Group transfer

–

Oxidation‐reduction

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Energetic Coupling -

Group Transfer

•

Transfer of a “high energy group”

onto or off of a  metabolic intermediate

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Energy Currency?

• Cells need to be able to store, transport and exchange energy

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Carbohydrates

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Fatty Acids and Lipids (Ch. 9)

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ATP and Metabolism

• One phosphate ester linked to ribose• 2 phosphoanhidrides• ATP can donate a phosphoryl group (Pi + ADP)

or a nucleotidyl group (AMP + PPi)• Transfer is usually to acceptor molecules

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* All cells contain pyrophosphatase so cellular [PPi] is very low

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Why ATP?

1. _

2. _

3. _

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Coupled Reactions/Role of ATP

• Overall ∆G must be negative for a reaction to proceed in a given direction

• Positive ∆G reactions need a driving energy source• Accomplished by coupling hydrolysis of ATP (or other

energy carrier) to reaction

X + Y XY

X + Y XYATP ADP + Pi

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Substrate Activation

•

What is “activation”?

•

Why do we need activation of metabolic  intermediates?

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Phosphoryl Group Transfer Potential?

• Hydrolysis of ATP provides significant energy

1.

2.

3.

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• The substrate or a side chain of the enzyme may be used as an intermediate acceptor of the phosphoryl group thereby transferring energy to the reaction.

Coupled Reactions/Role of ATP

1. X + ATP X-p + ADP2. X-p + Y + H2O XY + Pi + H+

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Phosphoryl group transfer in coupled reactions

Glutamate + NH4+ Glutamine + H2O ∆Gº’ = +14 kJ/mol

ATP ADP + Pi ∆Gº’ = -32 kJ/mol

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Phosphoryl group-transfer potential

High resonance energy56

Energetic coupling - Nucleotide Transfer

*

* Thioesters are high energy bonds, similar to phosphoanhydrideDrives fatty acid synthesis 57

Energetic Coupling –

Oxidation Reduction reactions

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Energy Coupling – Redox Reactions (ch. 6 & 10)

• Oxidation – Reduction (Redox) reactions involve the transfer of electrons from the reducing agent to an oxidizing agent (LEO says GER)– Oxidizing agent – accepts e- (and so is reduced)– Reducing agent – donates e- (and is oxidized)

• electrons can be transferred or “released”• In oxidation reactions released e- are transferred to

cofactor or coenzyme such as NAD+, NADP+, FMN, FAD or ubiquinone (Q)

Ared + Box Aox + Bred

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• Reduction Potential– A measure of thermodynamic activity – ability to accept e-

– “Standard” reduction reaction is H+ to H2 gas (0.0V)– Reduction potential is a measure of electromotive

force as determined using an electrochemical cell for a ‘half-reaction’

• Note: “redox potential” is often used but refers to the general ability of a molecule to accept or donate electrons (also called e- transfer potential)

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Reduction potential

• e- flow spontaneously from the more readily oxidized molecule to the more readily reduced molecule

• Std reduction potential for a given molecule is measured against H+ to H2

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Redox reactions

• Dehydrogenases– oxidation is accomplished by the removal of a

hydrogen atom (H – 1 proton, 1e-) or hydride ion (H+ -1 proton, 2e-)

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How much free energy is associated with reduction potential?

• Standard Reduction potential (Eº’) is a value determined in reference to H+/H2 in an electrochemical cell

• ** Eº’ is standardized at 10-7M [H+] (pH 7.0) where as ∆Gº’ is standardized at 1M [H+] (pH 0)

Eº’ is related to ∆Gº’ by:

∆Gº’ = -nF ∆Eº’

n = number of e- transferredF = faraday’s constant (96.48 kJ/ V∙mol)

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Free energy from the ability to reduce organic metabolites

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Standard reduction potentials for biological half-reactions

Eº’refers to the partial reaction as written:

Oxidized + e- → Reduced

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Using activated energy carriers

• During a reaction e- are transferred from metabolites to energy carriers such as NAD+

• NADH (reduced form) then becomes a source of e- in other redox reactions

• Oxidation of NADH and reduction of O2 produces a free energy change during membrane-associated electron transport and the energy is recovered in ATP synthesis

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Activated Energy Carriers in Redox reactions

NAD

FAD

2e- at a single reaction site

2e- at separate reaction sites

*

*

*

*

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Stable intermediates of quinones

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Reduction Potential and Free Energy

Next Class:

•

How does reduction potential relate to Gibbs Free  energy?

•

What equations are used to translate reduction  potential (E) into free energy (G)?

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Carbohydrate pathways

• Carbon Fixation, Calvin cycle• Glycolysis • Gluconeogenesis• Glycogen metabolism• Pentose phosphate pathway• Citric acid cycle

• Hormonal and feedback regulation

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• Fatty acid synthesis

• Lipid formation– TAGs, phospholipids, eicosanoids, ether lipids,

sphingolipids, cholesterol

• Fatty acid oxidation• Hormone regulation and lipid mobilization

Fatty Acids and Lipid Metabolism (Ch. 16)

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Supplemental Reading

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Next Class

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