BIOC13: Bioenergetics & Metabolism

Prof.MichelleAarts maarts@utsc.utoronto.ca OfficeHours:Tuesdays2 4pm inSW525 Contactmebyemail: forappointmentsoutsideofficehours 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)

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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Fundamentals of Biochemistry 2nd

Voet, Voet & Pratt (Wiley)

Biochemistry: The Molecular Basis of Life

McKee & McKee

Publisher websites
Pearson-Prentice Hall Wiley whfreeman

Posted on the Intranet

Syllabus&Courseinformation TakeHomeQuestions
CanyouanswerthesequestionsforeachTopic that wecoverinclass?

Lecturetopics(1 9) Supplementalreading Webresources Problemsets&answers

Evaluations!
Quizzes (4) Jan26th,Feb16th,March16th,April6th together 20% of final grade 5 10 questions (MC and written) Midterm 30% of final grade MC, fillintheblanks, short answer Final Exam 50% of final grade (cumulative content) MC, fillintheblanks, 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)

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

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

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?

Its 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) Whats 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 2003

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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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How are Metabolic Pathways Regulated?

A E1 B E2 C E3 D E4 P

Regulation of metabolic pathways occurs at the level of rate limiting enzymes 1. Allosteric Control 2. Covalent modification 3. Substrate cycles 4. Enzyme expression (genetic/degradation)
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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 Total entropy (disorder) of a system and its surroundings always increases
potential energy likelihood that 2 atoms will react with one another

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

surroundings

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Gibbs Free Energy Change (G)

G = H - TS A measure of the energy available from a reaction Standard Gibbs Free Energy Change (G)

Actual Gibbs Free Energy change (G)

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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+BC+D Reactions at equilibrium (no net change) have a G approaching zero

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Gibbs Free Energy

Derivationoftheequationsandhowtousethem nextclass

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The Standard Gibbs free energy difference (G) 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 G 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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Metabolic Flux and Equilibrium 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

G positive

G negative

G ~ 0
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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
Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases

Reaction Catalysed
Oxidation-reduction Move functional groups Hydrolysis Eliminate group & form double bond Isomerization Bond formation (ATP coupled)
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Some enzymes need cofactors

Aminoacidsidechainsmay participateinreaction Smallmolecules(Mg2+,Zn2+) stabilizesubstratebinding Coenzymes: Maybeaddedtosubstrate (coenzymeA) Soluble(ATP,NAD+) Boundprostheticgroups (FAD,FMN,biotin,

Coenzymes and Cofactors can be derived from Vitamins

GenerallyB Vitamins

Enzymes require cofactors (many derived from vitamins)

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Activated carriers or Co-factors

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Coenzyme A

carboxybiotin

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Metabolic Pathways - Energetic Coupling

Athermodynamicallyunfavorablereaction(+Gor Keq thatfavorssubstrates)canbedrivenforwardby couplingtoafavorablereaction 1 2 3

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Serial Coupling

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Coupling 2 Reactions
2individualreactionsoccurinthesameenzyme activesite(2substrates,2products) Grouptransfer Oxidationreduction

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Energetic Coupling - Group Transfer

Transferofahighenergygroup ontooroffofa metabolicintermediate

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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

X+Y XY

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 ATP XY ADP + Pi

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Substrate Activation
Whatisactivation? Whydoweneedactivationofmetabolic intermediates?

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Phosphoryl Group Transfer Potential?

Hydrolysis of ATP provides significant energy 1. 2. 3.

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Coupled Reactions/Role of ATP 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.
1. 2. X + ATP X-p + Y + H2O X-p + ADP XY + Pi + H+

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Phosphoryl group transfer in coupled reactions

Glutamate + NH4+ ATP

Glutamine + H2O ADP + Pi

G = +14 kJ/mol G = -32 kJ/mol

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Phosphoryl group-transfer potential

High resonance energy

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Energetic coupling - Nucleotide Transfer

* Thioesters are high energy bonds, similar to phosphoanhydride

Drives fatty acid synthesis
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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) Ared + Box Aox + Bred

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)
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 Reduction Potential
A measure of thermodynamic activity ability to accept eStandard 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 (E) is a value determined in reference to H+/H2 in an electrochemical cell ** E is standardized at 10-7M [H+] (pH 7.0) where as G is standardized at 1M [H+] (pH 0) E is related to G by: G = -nF E n = number of e- transferred

F = faradays constant (96.48 kJ/ Vmol)

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Free energy from the ability to reduce organic metabolites

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Standard reduction potentials for biological half-reactions Erefers 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

2e- at a single reaction site * * * *

NAD

2e- at separate reaction sites

FAD

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Stable intermediates of quinones

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Reduction Potential and Free Energy

NextClass: HowdoesreductionpotentialrelatetoGibbsFree energy? Whatequationsareusedtotranslatereduction potential(E)intofreeenergy(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 Acids and Lipid Metabolism (Ch. 16) Fatty acid synthesis Lipid formation
TAGs, phospholipids, eicosanoids, ether lipids, sphingolipids, cholesterol

Fatty acid oxidation Hormone regulation and lipid mobilization

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Supplemental Reading

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Next Class

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