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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)