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Figure 2-84 Molecular Biology of the Cell( Garland Science 2008)

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Electrontransportdrivesthesynthesis

ofthemajorityoftheATPinmostcells

NADH&FADH2 Electronstransferredtoelectrontransport

chain

Longchainofspecializedelectronacceptor&donormolecules

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Figure 2-85 Molecular Biology of the Cell( Garland Science 2008)

Electronsto

lowerenergy

state

Tomolecular

O2

H

+

gradientforms

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Figure 2-86 Molecular Biology of the Cell( Garland Science 2008)

CompleteoxidaGonofamoleculeoflucoseH2O&CO230ATP

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Aminoacids&nucleoGdes

Nitrogencycle OnlyfewlivingorganismscanfixN EssenGaltobiosphere Vertebratesfromdietaryintake

Proteins&nucleicacidsBrokendowntoaminoacids&nucleoGdes

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Figure 2-87 Molecular Biology of the Cell( Garland Science 2008)

Synthesizedbyplants&

otherorganisms

EnergeGcallyexpensive

pathways

Lostduringthe

evoluGonofvertebrates

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nucleoGdes

Purines&Pyrimidinesfromglutamine,asparGcacidandglycine

Ribose&deoxyribosesfromglucose NoessenGalnucleoGdes

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Aminoacids

Canalsobeusedtogenerateenergy OxidizetoH2O&CO2 Nitrogenexcretedintheformofurea

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Sulfurmetabolism

Abundant Oxidizedformofsulfate Neededtobereducedtosulfide,S-2 Vertebratescannotreducesulfate Mustbetakenindiet RequiredforthebiosynthesisofMet,Cys,CoA

Iron-sulfurcentersareessenGalforelectrontransport

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Metabolism

isorganized

andregulated

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Figure 2-88 Molecular Biology of the Cell( Garland Science 2008)

Samemoleculeispartofamanydifferent

pathways

Pyruvateissubstratefor>6enzymes

Leadingitsconversiontoadifferentmetabolite

MorecomplicatedinmulGcellularorganisms

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Networkofcontrolmechanisms

Differentmetabolictraitindifferentcells Metabolicbalance PerturbaGons

Disease&drugtreatmentstarvaGon

MetabolicresponsetoStress

eneGc Environmental

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RegulaGonofmetabolicnetworks

modulaGngenzymaGcreacGonrates

AcGvityofthekeyenzyme ShorterGmescale

ConcentraGonofthekeyenzyme Ontheorderofminutes&hours

AcGvity&concentraGon

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RegulaGonofgeneexpressionCoarsecontrolMinutes/Hours

RegulaGonofenzymeacGvityFinetuningShorterGmescale

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FuncGonofcellsbasedonComplexnetworksofinteracGngchemical

reacGons

OrganizedinspaceandGme

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Basicfeatures

Intermediarymetabolism Availablerawmaterialsconvertedinto

energy

buildingblocks

ChemicalmachineryDynamicLawsofphysics&chemistry

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TwotypesofchemicaltransformaGon

Catabolicpathways Commonsubstratesbrokendownintometabolites

Anabolicpathways Synthesisofbiologicalmolecules

Linkedthroughasetofcarriers

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Keychemicalgroupsinmetabolismand

theircarriers

Phosphoryl Electrons OneCunit Methyl Acyl(twoCunits) Aldehyde Carbondioxide nucleoGdes

ATP,TP NADH,NADPH,

FADH2,FMNH2

Tetrahyrofolate 5-adenosylmethionine CoenzymeA,lipoamine Thiaminepyrophosphate BioGne NTPS

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Models

Fourlevelsofcomplexity

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Level1:Wholecelllevelmodel

InputsSubstratestakenintothecellConvertedintobuildingblocks

Vitalproducts Maintenance

growth

OutputsBiomass&metabolicby-products

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DescripGonofacellatlevel1

CoarsegrainedConsistsof

asimplesetofcoupledmass&energybalances Empiricallydeterminedyieldcoefficients

rowthkineGcsMonodgrowthmodel

ModelsareusefulforalimitedsetofspecificcondiGons

IndustrialfermentaGonprocesses

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Level2:Metabolicsectors

FinergrainedlookTwobasicsectors

CatabolismDegradaGonofsubstratesAsetof11metabolitescalledbiosyntheGcprecursors

AnabolismMonomersfromthesebiosyntheGcprecursors

ModelsatthislevelofcomplexityusefultodescribegeneGcallyengineeredorganisms

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Level3:Pathways

FinerresoluGon Pathways:importantroles Catabolismofmajormacromolecules

SubstratestakeninHydolyzedifnecessaryAcGvatedbyacofactorDegradedtoyieldenergyOtherproperGesstoredoncarriermolecules

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Modelsatthislevelofcomplexity

BasicfeaturesofmetabolismBasicchemicalprenciplessuchas

stochiometricstructure&kineGcregulaGon

Keymetabolicpools(e.g.Energycharge)Keyregulatoryenzymeswhichdetermines

howmassandenergyisdistributed

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Level4:IndividualreacGons

FinestlevelofdescripGon AllbiochemicaltransformaGonsinacell HTdata Stochiometricmatrices

HundredsofmetabolitesOver1000ofreacGons

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BiochemicalTransformaGons

ClassifiedbyEnzymecomission(E.C.)anumberassociatedwitheachreacGon

ThermodynamicrestricGons(physicochemicalconstraints)

definetheenergeGcallyfeasablereacGons&itsequilibrium

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genomescalemetabolicmodel

reconstrucGon

AllreacGonsoccurringinthecell

Biochemicaldata,Databases

enomics

DNA

sequence

homology

AnnotaGon

Physiology&indirectinformaGon

Insilicomodellingdata(inferredreacGons)

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Reliabilityofdifferentdatasources

Biochemistry(4) eneGcdata(3) enomics(2) Physiology&indirectinformaGon(1)

(gapanalysis) insilicomodelingdata(0)

(addiGonofinferredreacGons)

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Biochemicaldata

MostreliablesourceforthepresenceofareacGon

Stoichiometry Reversibleornot gene:glk Enzyme:lucokinase ReacGon:ATP+D-glucose=ADP+D-glucosephosphate EC:2.7.1.2

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E.C.numbers

SystemaGcallycharacterizeenzymaGcreacGons

AmbiguousandduplicatenamesSuccinatedehydrogenase(sdh)Fumaratereductase(frd)

transportreacGonsasimilarclasificaGonsystemwasalsodeveloped

(26)

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Proteindatabases

Swiss-ProtProteinorreacGonassignementoldstandartLiteraturereferencesSequencesFuncGonalassignement

TrEMBLnewentriesintoSwiss-Protthathavenotyetbeencurated

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ene-Protein-ReacGon(PR)

AssociaGons

OnetoonerelaGonshipbetweengenes,proteins&reacGons?

MulG-subunits MorethanonegenesforonereacGon

Fumaratereductase 4subunits frdA,frdB,frdC,frdD

OneenzymecancatalyzemorethanonereacGon(promiscuousenzymes)

TransketolaseI

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OxidaGonofpyruvatetoacetylCoAandCO 2by

pyruvatedehydrogenasecomplex

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PRassociaGons

Succinatedehydogenase

ene bo721bo722bo723bo724 PepGde sdhCsdhD sdhAsdh Protein Sdh ReacGon SUCD1i SUCD4

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D-xyloseABCtransporter

ene b3566b3567b3568 PepGde xylFxylxylH Protein xylFxylxylH ReacGon XYLabc

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lyceraldehyde3-phosphate

dehydrogenase

ene b1779b1416b1417 PepGde gapAgapC2gapC1 Protein apAapC ReacGon APD

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TwoaddiGonalissuesinreconstrucGon

BiomassproducGon BiomasscomposiGon Experimentallydetermined BiomasscomposiGonofacloselyrelatedspecies

Physiologicaldata FuncGonalstatesofthenetwork Reconstructednetworkcanreproducethephysiological

behaviourthatisexperimentallyobserved

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Twofundamentallydifferentdatasets

DataonindividualreacGons ComponenttypeinformaGon Boom-updata

DataonfuncGonalstates WholenetworktypeinformaGon Top-downdata MetabolicnetworksarefuncGonallyhierarchical BothdatatypesareimportantinreconstrucGonprocess

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Publiclyavailablegenome

databases

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KEGG

(KyotoEncyclopediaofenesand

enomes)

acollec+onofonlinedatabases maintainsfivemaindatabases KEAtlas KEPathway KEenes

KELigand KEBRITE

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BiohemicaldatafundamentaltobothcuraGngandexpandingnetwork

Notcomplete

NewexperimentsIteraGvemodelbuilduingmay

acceleratethebologicaldiscovery

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ReconstrucGon:iteraGveprocess

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enomescalemetabolicmodelsinyeast

ORF Metabolite MetabolicRxn.

Further

Reac+on Cellular

compartments

Frsteretal.,2003(iFF708) 708 584 1035+140 1175

(842)

3

Duarteetal.,2004(iND750)

(compartmentalizaGon)

750 646 1498

(1149)

8

Moetal.,2009(iMM904)

(extracellularmetabolome)

904 872 1412

(1050)

8

Kuepferetal.,2005(iLL672)

(removeddeadends)

672 636

(579+166)

1038

(745)

2

Nookaewetal.,2008(iIN800)

(Lipidmetabolism)

800 907 1446

(907)

4

Herrgardetal.,2008(consensus)

(yeast1.0)

832 813 1857

(962)

15

Dobsonetal.,2010(consensus+)

(Lipidmetabolism)(yeast4.0)

924 924

(1102)

16

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ManchesterJamboree,2008

AconsensusyeastmetabolicnetworkreconstrucGonobtainedfromacommunityapproachtosystemsbiology

Herrgardetal

NatureBiotechnology26(10)1155-1160,2008

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enomescalemetabolicmodelsinyeast

ORF Metabolite MetabolicRxn.

Further

Reac+on Cellular

compartments

Frsteretal.,2003(iFF708) 708 584 1035+140 1175

(842)

3

Duarteetal.,2004(iND750)

(compartmentalizaGon)

750 646 1498

(1149)

8

Moetal.,2009(iMM904)

(extracellularmetabolome)

904 872 1412

(1050)

8

Kuepferetal.,2005(iLL672)

(removeddeadends)

672 636

(579+166)

1038

(745)

2

Nookaewetal.,2008(iIN800)

(Lipidmetabolism)

800 907 1446

(907)

4

Herrgardetal.,2008(consensus)(yeast1.0)

832 813 1857(962)

15

Dobsonetal.,2010(consensus+)

(Lipidmetabolism)(yeast4.0)

924 924

(1102)

16

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35metabolicmodelshp://systemsbiology.ucsd.edu/

In_Silico_Organisms/Other_organisms

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INTERATION

Metabolicnetworks

donotoperateinisolaGon interactwithothercellularprocesses

TranscripGonalregulaGonSignalingnetworks

Fateofthecells(apoptosisormitosisdecidedthroughinteracGonsofsignaling&metabolicnetworks)

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Metabolic,regulatory&signalingnetworkshave

commoncomponents

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MetabolicNetworks

METABOLICMODELLINTECHNIQUES

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MetabolicNetwork

A B C E

D

Reaction Intermediate

Active reaction

Inactive reaction

Substrate Product

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MetabolicNetwork

A B C E

D SystemBoundary

Exchange flux

Internal fluxFluxTheproducGonorconsumpGonofmass

perunitareaperunitGme.

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Boehringer-Mannheim

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DynamicModellingMetabolicControlAnalysis(MCA)

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ReacGonnetworks

complexreacGonsrepresentedinamore

compactform

thestoichiometrymatrix

nreac?ons

mpar?cipa?ngmolecularspecies

thestoichiometrymatrixwillhavecorrespondingncolumnsandmrows.

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vsyn

StoichiometryMatrix

Flux vectorConcentrationvector

Vsyn=Ksyn[A]

V=V(E,C,P)

TypicallynonlinearfuncGons/invitrokineGcs

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Dynamicmassbalance

StoichiometryMatrix

Flux vectorConcentrationvector Problem

V=V(k1, k2,k3) is a function ofconcentration &several kinetic parameters.

it is very difficult determine kinetic parameters

experimentally.

not enough kinetic information in the literatureto construct the model.

Solution !

assume the network is at steady state.

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**Dynamicmassbalanceatsteadystate

1. What does steady state mean?

2. Is it biologically justifiable toassume it?

3. Most important question

The steady state approximation isgenerally valid because of fastequilibration of metabolite

concentrations (seconds) withrespect to the time scale of genetic

regulation (minutes) Segre

2002

Yes

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3. Why does the steady state assumption help us solve ourproblem?

Steady stateassumption

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Metabolicraphs

A

B

C

AB ene1

BC+D ene2

A+DE ene3

D

Eene1

ene3

ene3

ene3ene2ene2

ene2

Integratewith

EnzymeacGviGes

or

eneexpressionprofiles

or

Metaboliteprofiles

DifferenGallyacGvated/repressed

metabolicpathways

ene1

ene2

ene3

B

D

A

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NetworkRepresentaGon

raph List Matrix

B A

B D

B C

A D

A B C D

A 0 1 0 1

B 1 0 1 1

C 0 1 0 0

D 1 1 0 0

B

CA

D

Whichone?

1.Dimension2.Sparsity

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AdjacencyMatrix(A)

Binary,square,sparse,symmetric(!)B

CA

D

B

CA

D

A B C D

A 0 1 0 1

B 1 0 1 1

C 0 1 0 0

D1 1 0 0

A B C D

A 0 0 0 1

B 1 0 1 0

C 0 0 0 0

D 0 1 0 0

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MetabolicNetwork

Stoichiometricmatrix(S): Rows:metabolites Columns:reacGons

Metabolitegraph: Nodes:metabolites Links:reacGons Adjacencymatrix

A=binary(Sb*SbT)

ReacGongraph: Nodes:reacGons

Links:metabolites Adjacencymatrix

A=binary(SbT*Sb)