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

Electron transport drives the synthesis of the majority of the ATP in most cells
NADH & FADH2 Electrons transferred to electron transport chain Long chain of specialized electron acceptor & donor molecules

Electrons to lower energy state To molecular O2 H+ gradient forms

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

Complete oxidaGon of a molecule of Glucose H2O & CO2 30 ATP

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

Amino acids & nucleoGdes

Nitrogen cycle Only few living organisms can x N EssenGal to biosphere Vertebrates from dietary intake
Proteins & nucleic acids Broken down to amino acids & nucleoGdes

Synthesized by plants & other organisms EnergeGcally expensive pathways Lost during the evoluGon of vertebrates

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

nucleoGdes
Purines & Pyrimidines from glutamine, asparGc acid and glycine Ribose & deoxyriboses from glucose No essenGal nucleoGdes

Amino acids
Can also be used to generate energy Oxidize to H2O & CO2 Nitrogen excreted in the form of urea

Sulfur metabolism
Abundant Oxidized form of sulfate Needed to be reduced to sulde, S-2 Vertebrates can not reduce sulfate Must be taken in diet Required for the biosynthesis of Met, Cys, CoA Iron-sulfur centers are essenGal for electron transport

Metabolism is organized and regulated

 Same molecule is part of a many dierent pathways Pyruvate is substrate for >6 enzymes
Leading its conversion to a dierent metabolite

More complicated in mulGcellular organisms

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

Network of control mechanisms

Dierent metabolic trait in dierent cells Metabolic balance PerturbaGons
Disease & drug treatment starvaGon

Metabolic response to
Stress
GeneGc Environmental

RegulaGon of metabolic networks

modulaGng enzymaGc reacGon rates
AcGvity of the key enzyme
Shorter Gme scale

ConcentraGon of the key enzyme

On the order of minutes & hours

AcGvity & concentraGon

 RegulaGon of gene expression

Coarse control Minutes/Hours

RegulaGon of enzyme acGvity

Fine tuning Shorter Gme scale

 FuncGon of cells based on

Complex networks of interacGng chemical reacGons Organized in space and Gme

Basic features
Intermediary metabolism Available raw materials converted into
energy building blocks

Chemical machinery
Dynamic Laws of physics & chemistry

Two types of chemical transformaGon

Catabolic pathways

Common substrates broken down into metabolites

Anabolic pathways

Synthesis of biological molecules

Linked through a set of carriers

Key chemical groups in metabolism and their carriers

Phosphoryl Electrons One C unit Methyl Acyl (two C units) Aldehyde Carbondioxide nucleoGdes ATP,GTP NADH,NADPH, FADH2,FMNH2 Tetrahyrofolate 5-adenosylmethionine Coenzyme A, lipoamine Thiamine pyrophosphate BioGne NTPS

Models Four levels of complexity

Level 1: Whole cell level model

Inputs
Substrates taken into the cell Converted into building blocks Vital products
Maintenance growth

Outputs
Biomass & metabolic by-products

DescripGon of a cell at level 1

Coarse grained
Consists of
a simple set of coupled mass & energy balances Empirically determined yield coecients

Growth kineGcs
Monod growth model

Models are useful for a limited set of specic condiGons

Industrial fermentaGon processes

Level 2: Metabolic sectors

Finer grained look Two basic sectors Catabolism
DegradaGon of substrates A set of 11 metabolites called biosyntheGc precursors

Anabolism
Monomers from these biosyntheGc precursors

Models at this level of complexity useful to describe geneGcally engineered organisms

Level 3: Pathways
Finer resoluGon Pathways: important roles Catabolism of major macromolecules Substrates taken in Hydolyzed if necessary AcGvated by a cofactor Degraded to yield energy Other properGes stored on carrier molecules

Models at this level of complexity

Basic features of metabolism Basic chemical prenciples such as stochiometric structure & kineGc regulaGon Key metabolic pools (e.g.Energy charge) Key regulatory enzymes which determines how mass and energy is distributed

Level 4: Individual reacGons

Finest level of descripGon All biochemical transformaGons in a cell HT data Stochiometric matrices
Hundreds of metabolites Over 1000 of reacGons

Biochemical TransformaGons
Classied by Enzyme comission (E.C.) a number associated with each reacGon Thermodynamic restricGons (physicochemical constraints) dene the energeGcally feasable reacGons & its equilibrium

 genome scale metabolic model reconstrucGon

All reacGons occurring in the cell Biochemical data , Databases Genomics DNA sequence homology AnnotaGon

Physiology & indirect informaGon In silico modelling data (inferred reacGons)

Reliability of dierent data sources

Biochemistry (4) GeneGc data (3) Genomics (2) Physiology & indirect informaGon (1)
(gap analysis) (addiGon of inferred reacGons)

in silico modeling data (0)

Biochemical data
Most reliable source for the presence of a reacGon Stoichiometry Reversible or not gene : glk Enzyme : Glucokinase ReacGon : ATP+D-glucose=ADP+D-glucose phosphate EC : 2.7.1.2

E.C. numbers
SystemaGcally characterize enzymaGc reacGons Ambiguous and duplicate names
Succinate dehydrogenase (sdh) Fumarate reductase (frd)

transport reacGons
a similar clasicaGon system was also developed (26)

Protein databases
Swiss-Prot
Protein or reacGon assignement Gold standart Literature references Sequences FuncGonal assignement

TrEMBL

new entries into Swiss-Prot that have not yet been curated

Gene-Protein-ReacGon (GPR) AssociaGons

One to one relaGonship between genes, proteins & reacGons ? MulG-subunits More than one genes for one reacGon
Fumarate reductase
4 subunits frdA, frdB, frdC, frdD

One enzyme can catalyze more than one reacGon (promiscuous enzymes)
Transketolase I

OxidaGon of pyruvate to acetyl CoA and CO2 by pyruvate dehydrogenase complex

GPR associaGons
Succinate dehydogenase
Gene PepGde Protein ReacGon

bo721 bo722 bo723 bo724 sdhC sdhD sdhA sdh Sdh SUCD1i SUCD4

D-xylose ABC transporter

Gene b3566 b3567 b3568 PepGde xylF xylG xylH Protein xylF xylG xylH ReacGon XYLabc

Glyceraldehyde 3-phosphate dehydrogenase

Gene b1779 b1416 b1417 PepGde gapA gapC2 gapC1 Protein GapA GapC ReacGon GAPD

Two addiGonal issues in reconstrucGon

Biomass producGon
Biomass composiGon Experimentally determined Biomass composiGon of a closely related species

Physiological data
FuncGonal states of the network Reconstructed network can reproduce the physiological behaviour that is experimentally observed

Two fundamentally dierent data sets

Data on individual reacGons
Component type informaGon Bomom-up data

Data on funcGonal states

Whole network type informaGon Top-down data Metabolic networks are funcGonally hierarchical Both data types are important in reconstrucGon process

Publicly available genome databases

KEGG (Kyoto Encyclopedia of Genes and Genomes)

a collec+on of online databases maintains ve main databases KEGG Atlas KEGG Pathway KEGG Genes KEGG Ligand KEGG BRITE

Biohemical data fundamental to both curaGng and expanding network Not complete New experiments IteraGve model builduing may accelerate the bological discovery

ReconstrucGon : iteraGve process

Genome scale metabolic models in yeast

ORF Metabolite Metabolic Rxn. + Further Reac+on Cellular compartments

Frster et al., 2003 (iFF708)

708

584

1035 + 140

1175 (842) 1498 (1149) 1412 (1050) 1038 (745) 1446 (907) 1857 (962)

Duarte et al., 2004 (iND750) (compartmentalizaGon) Mo et al., 2009 (iMM904) (extracellular metabolome) Kuepfer et al., 2005 (iLL672) (removed dead ends) Nookaew et al., 2008 (iIN800) (Lipid metabolism) Herrgard et al., 2008 (consensus) (yeast 1.0) Dobson et al., 2010 (consensus+) (Lipid metabolism) (yeast 4.0)

750

646

904

872

672

636

(579 + 166)

800

907

832

813

15

924

924

(1102)

16

 Manchester Jamboree, 2008

A consensus yeast metabolic network reconstrucGon obtained from a community approach to systems biology
Herrgard et al Nature Biotechnology 26(10)1155-1160,2008

 Genome scale metabolic models in yeast

ORF Metabolite Metabolic Rxn. + Further 1035 + 140 Reac+on Cellular compartments 3 8 8 2 4 15 16

Frster et al., 2003 (iFF708) Duarte et al., 2004 (iND750) (compartmentalizaGon) Mo et al., 2009 (iMM904) (extracellular metabolome) Kuepfer et al., 2005 (iLL672) (removed dead ends) Nookaew et al., 2008 (iIN800) (Lipid metabolism) Herrgard et al., 2008 (consensus) (yeast 1.0) Dobson et al., 2010 (consensus+) (Lipid metabolism) (yeast 4.0)

708 750 904 672 800 832 924

584 646 872 636 907 813 924

1175 (842) 1498 (1149) 1412 (1050)

(579 + 166)

1038 (745) 1446 (907) 1857 (962) (1102)

 35 metabolic models
hmp://systemsbiology.ucsd.edu/ In_Silico_Organisms/Other_organisms

INTEGRATION
Metabolic networks do not operate in isolaGon interact with other cellular processes
TranscripGonal regulaGon Signaling networks
Fate of the cells ( apoptosis or mitosis decided through interacGons of signaling & metabolic networks)

Metabolic, regulatory & signaling networks have common components

Metabolic Networks
METABOLIC MODELLING TECHNIQUES

Metabolic Network
Reaction A B Intermediate C E

Substrate

Product

Active reaction Inactive reaction

Metabolic Network
Exchange flux A B C E System Boundary

D Internal flux

Flux The producGon or consumpGon of mass per unit area per unit Gme.

Boehringer-Mannheim

 Dynamic Modelling Metabolic Control Analysis (MCA)

ReacGon networks
complex reacGons represented in a more compact form the stoichiometry matrix n reac?ons m par?cipa?ng molecular species the stoichiometry matrix will have corresponding n columns and m rows.

Concentration vector

Stoichiometry Flux vector Matrix

vsyn

Vsyn = Ksyn [A]

V = V(E, C, P) Typically nonlinear funcGons/ in vitro kineGcs

Dynamic mass balance

Concentration vector Stoichiometry Flux vector Matrix

Problem
V=V(k1, k2,k3) is a function of concentration & several kinetic parameters. it is very difficult determine kinetic parameters experimentally. not enough kinetic information in the literature to construct the model.

Solution !
assume the network is at steady state.

 ** Dynamic mass balance at steady state

1. What does steady state mean?

2. Is it biologically justifiable to assume it?

The steady state approximation is generally valid because of fast equilibration of metabolite concentrations (seconds) with respect to the time scale of genetic regulation (minutes) Segre 2002 Yes

3. Most important question

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

Steady state assumption

Metabolic Graphs
A

Gene3
E

A B B C+D A+D E

Gene1 Gene2 Gene3

Gene1
B

Gene3 Gene3
D

Gene2 Gene2
C

Gene2

Gene1

B Gene2

Gene3

Integrate with Enzyme acGviGes or Gene expression proles or Metabolite proles DierenGally acGvated/repressed metabolic pathways

Network RepresentaGon
Graph
B
A

List
B B B A

A D C D

Matrix
A A B C D 0 1 0 1 B 1 0 1 1 C 0 1 0 0 D 1 1 0 0

C D

Which one? 1.Dimension 2.Sparsity

Adjacency Matrix (A)

Binary, square, sparse, symmetric (!)
B
A

B C
A

C D

D A B C A 0 1 0 B 1 0 1 C 0 1 0 D 1 1 0 D 1 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

Metabolic Network
Stoichiometric matrix (S): Rows: metabolites Columns: reacGons Metabolite graph: Nodes: metabolites Links: reacGons Adjacency matrix A = binary(Sb*SbT) ReacGon graph: Nodes: reacGons Links: metabolites Adjacency matrix A= binary(SbT*Sb)