1

mSSB UE2.3 Metabolic Engineering

Cellular metabolism and the main metabolic pathways

By
Ioana Popescu
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Cellular metabolism

Metabolite:
• a naturally occurring molecule typically under 1000 MW
Enzyme:
• any protein which catalyzes chemical reactions involving the small molecules
• (ribozyme = catalytic RNAs)
Transporter:
• a membrane bound protein which shuttles ions, small molecules or macromolecules
across membranes, into cells or out of cells.

Metabolism:
• complete set of biochemical reactions within a cell
• sum of processes involved in energy conversions in the cell
Metabolic pathways
• complex sequences of controlled chemical reactions

Enzyme Enzyme Enzyme Enzyme Enzyme

catalysis catalysis catalysis catalysis catalysis
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Cellular metabolism

Metabolite:
• a naturally occurring molecule typically under 1000 MW

Background information from organic chemistry:

• Elements
• Bonds
• Molecules
• Isotopes
• Ions
• Nomenclature
• Stereochemistry
4

Chemical components of a cell

59%

11% 4% 24%
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Cellular metabolism

Metabolite:
• a naturally occurring molecule typically under 1000 MW

Background information from organic chemistry:

• Elements
• Bonds
• Molecules
• Isotopes
• Ions
• Nomenclature
• Stereochemistry

Building blocks Larger units

of the cell of the cell
SUGARS POLYSACCHARIDES
FATTY ACIDS FATS, LIPIDS, MEMBRANES
AMINO ACIDS PROTEINS
NUCLEOTIDES NUCLEIC ACIDS

Alberts et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science (2002)
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Cellular metabolism

Metabolite:
• a naturally occurring molecule typically under 1000 MW
Enzyme:
• any protein which catalyzes chemical reactions involving the small molecules
• (ribozyme = catalytic RNAs)
Transporter:
• a membrane bound protein which shuttles ions, small molecules or macromolecules
across membranes, into cells or out of cells.

Metabolism:
• complete set of biochemical reactions within a cell
• sum of processes involved in energy conversions in the cell
Metabolic pathways
• complex sequences of controlled chemical reactions

Enzyme Enzyme Enzyme Enzyme Enzyme

catalysis catalysis catalysis catalysis catalysis
7

Cellular metabolism
Glycan Nucleotides Cofactors & vitamins

Other secondary
Amino acids
Sugars metabolites
Lipids
Energy
Terpenoids &
Polyketides

Other amino acids

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Catabolic and anabolic pathways in cellular metabolism

Energy Secondary metabolites

Reducing
power

food

Precursor Building blocks Macromolecules

metabolites • Amino acids
• Nucleotides
• Sugars & derivatives
• Lipids
Heat • …

Waste
• Fermentation metabolites
• CO2

Catabolism Anabolism
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Classification of organisms
Designation Description
Source of Carbon • Autotroph • CO2
• Heterotroph • Organic molecules (e.g. sugars)
Source of • Organotroph • Organic molecules (e.g. sugars)
Electrons • Lithotroph • Inorganic molecules (e.g. H2, H2O, H2S, NH3, S)
Source of Energy • Phototroph • Light
• Chemotroph • Oxidation of chemical compounds
• Mixotroph • Light & Oxidation of chemical compounds
Ability to use O2 • Strict/Obligate aerobe • Require O2 for respiration – cannot survive
for respiration & without O2
sensitivity to O2 • Strict/Obligate anaerobe • Cannot survive in the presence of O2
• Facultative • Can survive in the presence or absence of O2
aerobe/anaerobe
• Aerotolerant anaerobe • Do not respire O2, but are able to survive in
the presence of O2

Human: Heterotroph, Organotroph, Chemotroph, Strict/Obligate aerobe

E. coli: Heterotroph, Organotroph, Chemotroph, Facultative aerobe/anaerobe
S. cerevisiae: Heterotroph, Organotroph, Chemotroph, Facultative aerobe/anaerobe
Plants & algae: Autotroph, Lithotroph, Phototroph, Strict/Obligate aerobe
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Different organisms – different cellular metabolisms

Saccharomyces Homo
cerevisiae sapiens

Escherichia
coli
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Cell content

E. coli S. cerevisiae H. sapiens

Metabolites 3 755 16 042 114 100
Reactions 8 254 31 624 6 302
Pathways 1 336 9 595 49 029
Enzymes 1 204 909 2 126
Transporters 299 149 0
Proteins 1 549 1 259 5 779
Metabolite synonyms 37 982 27 947 333 312
Metabolites in reactions 3518 (93.7 %) 11 157 (69.5 %) 23 473 (20.6 %)
http://www.ecmdb.ca http://www.ymdb.ca http://www.hmdb.ca

Sajed et al. Nucleic Acids Jewison et al. Nucleic Wishart et al. Nucleic
Research (2016) 44, Acids Research (2012) 40, Acids Research (2013) 41,
D495-D501 D815-D820 D801-D807
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Central pathways Glycolysis

Glycolysis Glucose Glycolysis:
ATP
ADP • Oxidation of 1 Glucose (6C)
Glucose-6-P Biosynthesis to 2 Pyruvate (3C)
• Production of cellular
Fructose-6-P Biosynthesis
ATP energy sources:
ADP • 2 ATP
Fructose-1,6-bisP
• 2 NADH,H+
Dihydroxyacetone-P Glyceraldehyde-3-P Biosynthesis
• Supply of 6 precursor
NAD+ metabolites for biosynthesis
NADH,H+
1,3-bisphosphoglycerate • ΔG’°= – 74 kJ.mole-1
ADP
ATP
• ΔG’= – 98 kJ.mole-1
3-phosphoglycerate Biosynthesis

2-phosphoglycerate

Phosphoenolpyruvate Biosynthesis
ADP
ATP
Pyruvate Biosynthesis

Fermentation TCA cycle

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Central pathways Glycolysis

Glycolysis Glucose Trehalose Glycogen / starch Lactose
ATP
ADP
Glucose-6-P Glucose-1-P UDP-Glucose UDP-Gal Galactose
ADP ATP Manose-6-P Manose
Fructose-6-P Fructose Sucrose
ATP ATP
ADP ADP
Fructose-1,6-bisP Fructose-1-P
ADP ATP
Dihydroxyacetone-P Glyceraldehyde-3-P Glyceraldehyde
NAD+
NADH,H+
1,3-bisphosphoglycerate
ADP
ATP
3-phosphoglycerate

2-phosphoglycerate
Others mono, di, polysaccharides enter glycolysis
Phosphoenolpyruvate
ADP
ATP Diauxic growth:
Pyruvate multiple carbon sources are utilized sequentially

Fermentation TCA cycle

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Central pathways Pentose Phosphate pathway

Glycolysis Glucose Pentose Phosphate Pathway
ATP NADP+ NADPH,H+ NADP+ NADPH,H+
ADP
Glucose-6-P 6-phosphoglucuronate Ribulose-5-P + CO2

Fructose-6-P Biosynthesis Ribose-5-P Xylulose-5-P

ATP
ADP
Fructose-1,6-bisP Sedoheptulose-7-P Glyceraldehyde-3-P

Dihydroxyacetone-P Glyceraldehyde-3-P Fructose-6-P Erythrose-4-P

NAD+
NADH,H+
1,3-bisphosphoglycerate
ADP
ATP Biosynthesis
3-phosphoglycerate

2-phosphoglycerate Pentose Phosphate Pathway:

• Oxidation of 1 Glucose-6-P (6C) to
Phosphoenolpyruvate 1 Glyceraldehyde-3-P and 3 CO2
ADP
ATP • Production of cellular energy sources:
Pyruvate • 6 NADPH,H+ (per 1 Glucose-6-P)
• Supply of 2 precursor metabolites for
Fermentation TCA cycle biosynthesis
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Central pathways ED pathway

Glycolysis Glucose Entner–Doudoroff Pathway:
ATP NADP+ NADPH,H+
ADP • Only in some bacteria
Glucose-6-P 6-phosphoglucuronate (ex. E. coli), archeae and
Entamoeba histolyca,
Fructose-6-P 2-keto-3-deoxy- Aspergillus niger, Penicillum
ATP
ADP 6-phosphogluconate notatum
Fructose-1,6-bisP • Oxidation of 1 Glucose
(6C) to 2 Pyruvate (3C)
Dihydroxyacetone-P Glyceraldehyde-3-P
NAD+
• Production of cellular
NADH,H+ energy sources:
1,3-bisphosphoglycerate
ADP • 1 ATP
ATP
3-phosphoglycerate
• 1 NADH,H+
• 1 NADPH,H+
2-phosphoglycerate • Supply of 5 precursor
metabolites for
Phosphoenolpyruvate biosynthesis
ADP
ATP Entner–Doudoroff
Pyruvate Pathway

Fermentation TCA cycle

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Central pathways ED pathway

Glycolysis Glucose Entner–Doudoroff Pathway:
ATP NADP+ NADPH,H+
ADP • Only in some bacteria
Glucose-6-P 6-phosphoglucuronate (ex. E. coli), archeae and
Entamoeba histolyca,
2-keto-3-deoxy- Aspergillus niger, Penicillum
6-phosphogluconate notatum
• Oxidation of 1 Glucose
(6C) to 2 Pyruvate (3C)
Glyceraldehyde-3-P_
NAD+
• Production of cellular
NADH,H+ energy sources:
1,3-bisphosphoglycerate
ADP • 1 ATP
ATP
3-phosphoglycerate
• 1 NADH,H+
• 1 NADPH,H+
2-phosphoglycerate • Supply of 5 precursor
metabolites for
Phosphoenolpyruvate biosynthesis
ADP
ATP Entner–Doudoroff
Pyruvate Pathway

Fermentation TCA cycle

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Central pathways Metabolism of carbohydrates and carboxylic acids

Glycolysis Glucose Pentose Phosphate Pathway
ATP NADP+ NADPH,H+ NADP+ NADPH,H+
ADP
Glucose-6-P 6-phosphoglucuronate Ribulose-5-P + CO2

Fructose-6-P 2-keto-3-deoxy- Ribose-5-P Xylulose-5-P

ATP
ADP 6-phosphogluconate
Fructose-1,6-bisP Sedoheptulose-7-P Glyceraldehyde-3-P

Dihydroxyacetone-P Glyceraldehyde-3-P Fructose-6-P Erythrose-4-P

NAD+
NADH,H+
1,3-bisphosphoglycerate
ADP
ATP • Oxidation of Glucose (6C) to
3-phosphoglycerate
Pyruvate (3C)
2-phosphoglycerate
• Production of cellular
energy sources:
Phosphoenolpyruvate • ATP
ADP
ATP Entner–Doudoroff • NADH,H+
Pyruvate Pathway • NADPH,H+
• Supply of 8 precursor
Fermentation TCA cycle metabolites for biosynthesis
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Central pathways Fermentation

Glucose

Oxaloacetate Phosphoenolpyruvate
NADH,H+ ADP NADH NAD+ Lactobacillus sp., animals
NAD+ ATP
Malate Pyruvate Lactate
NAD+ NADH,H+
Fumarate Acetaldehyde Acetyl-CoA + Formate CO2 + H2
FADH2 NADH,H+
FAD NAD+
Succinate Ethanol Acetyl-P Acetoacetyl-CoA
ADP NADH,H+
Yeasts ATP NAD+
(ex. S. cerevisiae), Acetate β-hydroxy-butyryl-CoA Acetoacetate
some fungi
Crotonyl-CoA Acetone
NADH,H+ NADH,H+
Fermentation Butyryl-CoA
NAD+
Isopropanol
NAD+

• No external electron acceptors NADH,H

NAD+
+

• No respiratory pathway Butyryl-P Butyraldehyde

+
• NAD regeneration ADP NADH,H+
ATP NAD+
• Reduced products (acids & alcohols) Butyrate Butanol
• Poor energy yield (ATP) Clostridium beijerinckii
• ATP produced only by substrate-level phosphorylation
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Central pathways Fermentation

Glucose

Oxaloacetate Phosphoenolpyruvate
NADH,H+ ADP NADH NAD+
NAD+ ATP
Malate Pyruvate Lactate
NAD+ NADH,H+
Fumarate Acetaldehyde Acetyl-CoA + Formate CO2 + H2
FADH2 NADH,H+
FAD NAD+
Succinate Ethanol Acetyl-P Acetoacetyl-CoA
ADP NADH,H+
ATP NAD+
E. coli Acetate β-hydroxy-butyryl-CoA Acetoacetate

Crotonyl-CoA Acetone
NADH,H+ NADH,H+
Fermentation Butyryl-CoA
NAD+
Isopropanol
NAD+

• No external electron acceptors NADH,H

NAD+
+

• No respiratory pathway Butyryl-P Butyraldehyde

+
• NAD regeneration ADP NADH,H+
ATP NAD+
• Reduced products (acids & alcohols) Butyrate Butanol
• Poor energy yield (ATP)
• ATP produced only by substrate-level phosphorylation
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Central pathways Krebs cycle

TCA Cycle: Glucose

• Oxidation of 1 Acetyl-CoA to
2 CO2 Phosphoenolpyruvate
ADP ADP
• Production of cellular ATP ATP
Pyruvate Acetate + CO2
energy sources: NADH,H+ ATP
ATP
• 1 ATP NAD(P)H,H+
ATP NAD+
AMP
ADP
+ ADP Acetyl-CoA Acetyl-P
• 3 NADH,H NAD(P)+

• 1 FADH2
Biosynthesis Oxaloacetate Citrate Biosynthesis
• Supply of 4 precursor NADH,H+
NAD+
metabolites for Malate cis-aconitate
Krebs cycle
biosynthesis TCA cycle
• Operates: Fumarate Isocitrate
FADH2 NAD(P)+
• Completely only in FAD NAD(P)H,H+

the presence of an Succinate α-ketoglutarate Biosynthesis

ATP NAD+
external electron ADP NADH,H+
Succinyl-CoA
acceptor
Biosynthesis
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Central pathways Krebs cycle

TCA Cycle: Glucose

• Oxidation of 1 Acetyl-CoA to
2 CO2 Phosphoenolpyruvate
ADP ADP
• Production of cellular ATP ATP
Pyruvate Acetate + CO2
energy sources: NADH,H+ ATP
ATP
• 1 ATP NAD(P)H,H+
ATP NAD+
AMP
ADP
+ ADP Acetyl-CoA Acetyl-P
• 3 NADH,H NAD(P)+

• 1 FADH2
Biosynthesis Oxaloacetate Citrate Biosynthesis
• Supply of 4 precursor NADH,H+
NAD+
metabolites for Malate cis-aconitate
Krebs cycle
biosynthesis TCA cycle
• Operates: Fumarate Isocitrate
FADH2 NAD(P)+
• Completely only in FAD NAD(P)H,H+

the presence of an Succinate α-ketoglutarate Biosynthesis

ATP
external electron ADP
Succinyl-CoA
acceptor
• Partially in the absence Biosynthesis
of an external electron
acceptor
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Central pathways Respiration

External terminal electron acceptors:

Reduced A ox B red ... ox Z red Oxidized
Substrat e- acceptor
…..
.....
Oxidized Reduced
Substrat A red B ox … red Z ox e- acceptor

Type of respiration External terminal electron acceptors Reduced products

Aerobic respiration O2 H2O
Fumarate: HOOC-CH=CH-COOH Succinate: HOOC-CH2-CH2-COOH
Trimethylamine N-oxide: (CH3)3-N®O Trimethylamine: (CH3)3-N
Anaerobic respiration
S, S2O32-, SO42- H2S
NO3-, NO2-, NO N2
Fe3+ Fe2+

• A H+ (electrochemical) gradient is produced across a membrane = proton-motrice force

• Flow of H+ down the gradient (across the membrane) through the H+ channel of ATP
synthase: ATP synthesis = oxidative phosphorylation
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Central pathways Eukaryotic aerobic respiration

• Oxidative phosphorylation
• Mitochondria – internal membrane
P/O:
• NADH+H+ + ½ 02 ® NAD+ + H2O G°’ = -218 kJ.mol-1 Þ ~0,96 – 3 ATP
15 to 38 ATP /
• FADH2 + ½ 02 ® FAD + H2O G°’ = -165 kJ.mol-1 Þ ~0,88 – 2 ATP
Glucose

H+ H+ H+ H+ H+ H+ H+ H+ H+ H+

I II III IV ATP
synthase
H+
CoQ CoQ Cyt c

½ O2 H2O H+
Fumarate
NADH+H+ Succinate
NAD+
ADP + Pi ATP
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Central pathways Prokaryotic respiration (bacteria & archaea)

• Cytoplasmic membrane
• Complexity – ability to adapt to different
• Growth conditions
• Electron donors: NADH, NADPH, FADH2, organic substances, H2, NH3, NO2-, S, S2-, Fe2+
• Electron acceptors:
• organic compounds (fumarate, dimethyl sulfoxide, trimethylamine N-oxide)
• inorganic compounds (O2, NO3-, NO2-, NO, ClO3-, ClO4-, S, S2O32-, SO42-, SeO42-,
AsO43-, CO2, oxidized manganese ions, gold, Fe3+)

e- Donor e- Donor e- Donor

¯ ¯ ¯
Dehydrogenase ® Quinones ® Cytochromes
¯ ¯
Reductases Terminal oxidases
¯ ¯
e- Acceptor e- Acceptor
Anaerobic Aerobic
conditions conditions
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Central pathways E. coli aerobic respiration

NADH:ubiquinone NADH:ubiquinone Cytochrome bo Cytochrome bd-I

oxidoreductase I oxidoreductase II Terminal oxidase Terminal oxidase
• used preferentially • expressed at • expressed at
in aerobic and high O2 levels low O2 levels
nitrate respiration • Low Km for O2

4 H+ 2 H+

2 H+ 2 H+

CoQ CoQ CoQ CoQ

CoQH2 CoQH2 CoQH2 CoQH2

NADH,H+ NAD+ NADH,H+ NAD+ ½ O2 + 2 H+ H2O ½ O2 + 2 H+ H2O

4 H+ 2 H+

Used both during glucose limited

aerobic growth
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Central pathways

Metabolism of carbohydrates and carboxylic acids

• Oxidation of 1 Glucose to
• 6 CO2 (Complete oxidation)
• Various fermentation products (Partial oxidation)
• Production of cellular energy sources:
• Up to 38 ATP / glucose
• Reducing power (NADH, NADPH, …)
• Tight regulation:
• Redox potential
• Energy charge
• Presence of terminal e- acceptors
• Growth substrate
• Amount
• Type
• Catabolite repression
• Diauxic growth: multiple carbon sources are utilized sequentially
• Allosteric modulation of enzymes
• Covalent modifications of enzymes (e.g. phosphorylation)
• Transcriptional control
• Compartmentalization & transport
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Central pathways – Precursor metabolites – Building blocks

Metabolism of carbohydrates and carboxylic acids

• Supply of 12 major precursor metabolites for biosynthesis
• Used with ATP and NAD(P)H for the biosynthesis of cellular building blocks

Precursor metabolites Pathway Amount required for Building blocks produced

biosynthesis of E. coli
Glucose-6-P Glycolysis 205 µmol / g cell NDP-glucose
Fructose-6-P Glycolysis 71 µmol / g cell NDP-mannose, NDP-N-acetylglucoseamine, …
Glyceraldehyde-3-P Isoprenoids
Glycolysis 129 µmol / g cell
Dihydroxyacetone-P Glycerol-P (& lipids)
3-Phosphoglycerate Glycolysis 1496 µmol / g cell Gly, Ser, Cys, nucleotides (purine)
Phosphoenolpyruvate Glycolysis 519 µmol / g cell Phe, Tyr, Trp
Pyruvate Glycolysis 2833 µmol / g cell Ala, Val, Leu, Ile, Lys, Isoprenoids
Ribose-5-P Pentose phosphate 898 µmol / g cell His, nucleotides (pentose)
Erythrose-4-P Pentose phosphate 361 µmol / g cell Phe, Tyr, Trp
Acetyl-CoA TCA cycle 3748 µmol / g cell Leu, Fatty acids, Isoprenoids
α-Ketoglutarate TCA cycle 1079 µmol / g cell Glu, Gln, Pro, Arg, nucleotides (purine)
Oxaloacetate TCA cycle 1787 µmol / g cell Asp, Asn, Lys, Thr, Met, Ile, nucleotides
Succinyl-CoA TCA cycle Met, Lys, tetrapyrroles (ex. Heme)
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Precursor metabolites – Building blocks – Macromolecules

Metabolism of carbohydrates and carboxylic acids

• Supply of 12 major precursor metabolites for biosynthesis
• Used with ATP and NAD(P)H for the biosynthesis of cellular building blocks
• Polymerization of building blocks
Precursor metabolites Building blocks Macromolecules

Glucose-6-P
Sugars (activated) Polysaccharides
Fructose-6-P
Glyceraldehyde-3-P
Dihydroxyacetone-P Amino acids Proteins & Peptides
3-Phosphoglycerate
Phosphoenolpyruvate
Nucleotides Peptidoglycan
Pyruvate
Ribose-5-P
Fatty acids
Erythrose-4-P Nucleic acids (DNA, RNA)
Acetyl-CoA
Isoprenoids
α-Ketoglutarate
Oxaloacetate Glycerol-P Lipids
Succinyl-CoA Tetrapyrroles
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Isoprenoids / Terpenoids over 25 000 known compounds

Mevalonate MEP/DXP Core cellular metabolites
pathway pathway
FPP H

b ol i tes n

meta
Ubiquinone Sterols

con dary
Se Lycopene

Carotenoids

ß-Carotene
Valencene

Artemisinin
Nootkatone Limonene Pinene Myrcene
Flavors
Fuels
Pharmaceuticals
Farnesene

Bisabolene

FPP Farnesol
Taxol
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Secondary metabolism

Not essential for Bioactive molecules

• Growth • Pharmaceuticals
• Respiration • Antibacterials
• Development • Antifungals
• Reproduction • Antitumor
• Antihelminthic
Beneficial for the producer: • Antiviral
• Improve survival fitness • Herbicidal
• Bactericides or fungicides • Insecticidal
• Specific receptors • Immunosupressors
¯
Produced at low titers Interesting for humans
¯
Complex organic compounds Candidates for metabolic engineering
• Difficult to synthetize
• Difficult to derivatize Main classes
• Isoprenoids / Terpenoids
• Alkaloids
• Flavonoids
• Non ribosomal peptides
• Polyketides
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Secondary metabolism plants

Primary Primary
metabolites metabolites

Wilson & Roberts. Current Opinion in Biotechnology (2014) 26, 174–182

32

Secondary metabolism fungi

Non-ribosomal peptides

Ex. penicillin production by

Primary
Penicillium chrysogenum
metabolites
33

Secondary metabolism bacteria

Polyketides Primary
metabolites
Ex. Erythromycin
production by
Saccharopolyspora
erythraea

Weissman & Leadlay. Nature Reviews Microbiology (2005) 3, 925-936

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Producing sugars Gluconeogenesis

Gluconeo Glucose Gluconeogenesis:
genesis • synthesis of glucose from
Glucose-6-P noncarbohydrate precursors
• Pyruvate
Fructose-6-P Glycerol-P
ATP • Lactate
ADP • Glycerol
Fructose-1,6-bisP NADH,H+ NAD+ Glycerol
• Amino acids (all but Lys, Leu)
Dihydroxyacetone-P Glyceraldehyde-3-P
• From pyruvate:
NAD+ • 7 glycolytic enzymes
NADH,H+
1,3-bisphosphoglycerate • 4 enzymes to bypass the 3
ADP
ATP
irreversible steps of
3-phosphoglycerate glycolysis
• Cost:
2-phosphoglycerate • 6 ATP
• 2 NADH,H+
Asp, Asn
Phosphoenolpyruvate
ADP Fumarate Tyr, Phe
ATP Krebs cycle
Oxaloacetate Succinyl-CoA Met, Val, Ile, Thr
ADP NADH,H+ NAD+ α-ketoglutatate Glu, Gln, Pro, His, Arg
ATP
Pyruvate Lactate
Gly, Ala, Ser, Cys, Trp
35

Producing sugars Glyoxylate bypass

Glucose

Phosphoenolpyruvate
ADP ADP
ATP ATP
Pyruvate
NADH,H+
ATP NAD+
ADP Acetyl-CoA Fatty acids

Oxaloacetate Citrate
NADH,H+
NAD+
Malate cis-aconitate
Glyoxylate

Fumarate Isocitrate
FADH2 NAD(P)+
FAD NAD(P)H,H+
Glyoxylate by-pass: Succinate α-ketoglutarate
ATP NAD+
• Germinating seeds of plants ADP NADH,H+
• Some bacteria (ex. E. coli) Succinyl-CoA
• Some invertebrates
• Glyoxysomes of eucaryotes
• Allows grow on acetate and fatty acids: 2 Acetyl-CoA ® 1 Succinate
• Supply of precursor metabolites for biosynthesis
36

Producing sugars Photosynthesis

• Autotroph organisms
• CO2 assimilation / CO2 fixation / carbon fixation
• Phototroph
• Energy = light

H2O O2

Light
reactions

NADP+ NADPH,H+
6 CO2 + 6 H2O ® C6H12O6 + 6 O2
ADP + Pi ATP

Carbon fixation
reactions

Carbohydrate CO2
37

Producing sugars Photosynthesis

Light reactions
• e- flow: from H2O to NADP+
• A H+ (electrochemical) gradient is produced across a membrane = proton-motrice force
• Flow of H+ down the gradient (across the membrane) through the H+ channel of ATP
synthase: ATP synthesis = oxidative phosphorylation

2 H2O + 2 NADP+ + 3 ADP + 3 Pi

¯
O2 + 2 NADPH,H+ + 3 ATP
38

Producing sugars Photosynthesis

Calvin cycle
• The dark reactions of photosynthesis
• In stoma of chloroplasts

3 CO2 + 9 ATP + 6 NADPH,H+ + 5 H2O

¯
Glyceraldehyde-3-P + 9 ADP + 8 Pi + 6 NADP+
39

Producing sugars Reverse/reductive TCA cycle

Arnon-Buchanan cycle: Glucose

Ex. Chlorobium thiosulfatophilum
• Used by some autotrophic • Photosynthetic green sulfur
anaerobic and Phosphoenolpyruvate
bacterium
AMP
microaerobic bacteria and ATP • Anaerobic
Pyruvate
archaea for CO2 Fdox
• Inorganic medium:
assimilation CO2 CO2
Fdred • sulfide and thiosulfate
Acetyl-CoA as e- donors
• two ferredoxin-linked ADP
(Fd) CO2-fixation reactions ATP • CO2 as an obligatory
Oxaloacetate Citrate carbon source = Strictly
are O2 sensitive autotrophic
NADPH,H+
NADP+
Malate cis-aconitate
rTCA cycle
Fumarate Isocitrate
FADH2 NADP+
FAD NADPH,H+ CO2

Succinate ATP Fdox

α-ketoglutarate
ADP Fdred
Succinyl-CoA
CO2

Berg. Applied and Environmental Microbiology (2011) 77, 1925-1936

 40

Producing sugars Reductive Acetyl-CoA pathway

Wood-Ljungdahl pathway:
• Used by some autotrophic anaerobic bacteria and archaea for CO2 assimilation
• Acetogenic bacteria
• Methanogenic archeae • High energetic efficiency
• … • Limited to a few ecological niches
• One CO2 ® CH3 • Anaerobiosis
• One CO2 ® CO • Metals: Mo or W, Co, Ni, and Fe
• Cofactors: tetrahydropterin and cobalamin

(tetrahydrofolate)

(methanofuran)

CO
dehydrogenase/acetyl
-CoA synthase

(tetrahydropterin)

Berg. Applied and Environmental Microbiology (2011) 77, 1925-1936

41

Producing sugars 3-hydroxypropionate bi-cycle

Fuchs-Holo bi-cycle
• Used only by green nonsulfur bacteria of Chloroflexaceae family
• Phototroph, chemotroph & mixotroph
• Autotroph & heterotroph
• Anoxygenic
• Co-assimilation of CO2 & numerous compounds (e.g., fermentation products) = mixotrophy
• High energy requirements
• 7 ATP for Acetyl-CoA to Pyruvate
• No oxygen-sensitive steps

Berg. Applied and Environmental Microbiology (2011) 77, 1925-1936

42

Producing sugars 4-hydroxybutyrate cycles

4-hydroxybutyrate cycles
• Used only by the hyperthermophilic archaea of Crenarchaeota family
• dicarboxylate/4-hydroxybutyrate • 3-hydroxypropionate/4-
(DC/HB) cycle hydroxybutyrate (HP/HB) cycle
• Desulfurococcales and Thermoproteales • Sulfolobales
• Anaerobic • Aerobic
• 5 ATP • 9 ATP

CD/HB cycle HP/HB cycle

Berg. Applied and Environmental Microbiology (2011) 77, 1925-1936

43

Transport & Metabolism: linked processes in the cell

Ex: the lac operon

Absence of Inducer

Presence of Inducer

Pierce. Genetics (2nd Ed.) Freeman & Co (2005)

44

Transport across membranes

Membranes:
• Semi-permeable structure • Phospholipid bilayer :
• Dynamic • Esters of glycerol + fatty acids
• Barrier with selective permeability + phosphate
• Controlled entry & exit • Eukaryotes
• Entry: • Bacteria
• Nutriments • Phospholipid monolayer :
• Signaling substances • Tetraethers of glycerol + fatty
• Nucleic acids alcohols (+ phosphate esters)
• Metal ions • Archaea
• Exit:
• Products generated by metabolism • Other lipids – for rigidity
• Secreted substances • Sterols
• Lipids • All eukaryotes
• Carbohydrates • Bacteria: methanotrophs
• Proteins & mycoplasma
• Nucleic acids • Hopanes
• Toxic substances • Bacteria
• Metal ions
• Damaged ® cell death • Proteins = 75 % of membrane mass
45

Transport across membranes Bacteria

46

Transport across membranes Eucaryotes

Saccharomyces cerevisiae
47

Transport classification: TC system

Passive transport:
• No energy required
• Occurs only in the downhill direction of concentration gradient

Facilitated diffusion:

Diffusion Pores & channels Uniporters Symporters Antiporters

H2O , O2 , CO2 , TC#1 TC#2 TC#2 TC#2
non polar organic
molecules
http://www.tcdb.org
48

Transport classification: TC system

Active transport: Group translocators:

• Primary energy required • substrate is modified during
• Chemical (ATP) the transport process
• Electrical
• Solar
• Occurs against a concentration gradient Glucose

Phosphoenolpyruvate

ATP Pyruvate Glucose-6P

Primary Active Transporters Group translocators

TC#3 TC#4

TC#5: Transmembrane electron flow systems http://www.tcdb.org

49

Kinetics of Transport processes Diffusion

Movement of Sx is described by its flux JSX:

• number of moles of Sx crossing a unit area of cell membrane per unit of time (moles.cm-2s-1)
• Depends on :
• permeability coefficient PSX:
• Sx solubility in lipids Fick’s Law:
• Sx diffusion coefficient in lipids JSX = PSX [SX]0 – PSX [SX]i
• Membrane thickness influx efflux
• Gradient across membrane
• Decomposed into:
• Unidirectional influx JSX0®i – proportional to the outside concentration
• Unidirectional efflux JSXi®0 – proportional to the inside concentration
• at equilibrium [SX]0 = [SX]i - if Sx is not charged

If Sx is charged: voltage difference across membrane should be considered

Electrochemical energy: R = gas constant = 8,315 J.mol-1K-1

T = temperature °K
ΔμSX = RT ln ([SX]i /[SX]0) + zXF (Ψi – Ψ0) zX = charge of SX
F = Faraday constant = 96500 C
Ψi – Ψ0 = voltage difference across membrane
50

Kinetics of Transport processes

Carrier mediated transport + diffusion

Rate of transport

Carrier mediated transport Carrier mediated transport:

• Affinity
• Specificity
Diffusion • Energy availability
• Regulation
• Localization
Major conformational changes
Substrate concentration
Steps: bind – transport – release
… like enzymes

Michaelis-Menten transport kinetics

[SX]0
V = Vmax
Km + [SX]0
51

Different organisms – different cellular metabolisms

52

Cellular metabolism

Metabolite:
• a naturally occurring molecule typically under 1000 MW
Enzyme:
• any protein which catalyzes chemical reactions involving the small molecules
• (ribozyme = catalytic RNAs)
Transporter:
• a membrane bound protein which shuttles ions, small molecules or macromolecules
across membranes, into cells or out of cells.

Metabolism:
• complete set of biochemical reactions within a cell
• sum of processes involved in energy conversions in the cell
Metabolic pathways
• complex sequences of controlled chemical reactions

Enzyme Enzyme Enzyme Enzyme Enzyme

catalysis catalysis catalysis catalysis catalysis
53

Cellular metabolism

Enzyme:
• Any protein which catalyzes chemical reactions involving the small molecules
• Catalysis = acceleration of chemical reactions – useful time scale
• Efficiency
• Selectivity
• Specificity / Promiscuity Enzyme
• Mild conditions of temperature and pH catalysis
• Regulation – for coordination of different metabolic pathways

History:
• 1752: René Antoine Ferchault de Réaumur - first recognition and description biological
catalysis = digestion of meat by secretions of stomach
• 1877: Wilhelm Kühne - first used the term enzyme = the unformed or not organized
ferments, whose action can occur without the presence of organisms and outside of the
same
• 1926: James B. Sumner - showed that the enzyme urease was a pure protein
• 1982: Thomas R. Cech and Sidney Altma - discovery of ribozymes (Nobel Prize in
Chemistry 1989)
54

Enzymes

• Primary, secondary, tertiary, quaternary structure = essential for activity

• Some require post-translational modifications (phosphorylation, glycosylation, …)
• Some require cofactors: • Prostetic group =
• Inorganic ions: Na, K, Mg, Cu, Fe, Mn, Mo, Zn, Ni, Se, … cofactor very tightly or
• Complex organic molecules = coenzymes even covalently bound

Coenzyme Dietary precursor in mammals

Thiamine pyrophosphate Vitamin B1 = thiamine
Flavin adenine dinucleotide (FAD) Vitamin B2 = riboflavin
FAD
Nicotinamide ademine dinucleotide (NAD) Vitamin B3 = nicotinic acid (niacin)
Coenzyme A Vitamin B5 = panthothenic acid
Pyridoxal phosphate Vitamin B6 = pyridoxine
Biocytin Vitamin B8 (H) = biotin
Tetrahydropholate Vitamin B9 (M) = folic acid
5’deoxyadenosylcobalamin Vitamin B12 = cobalamin
NAD+
Ascorbate Vitamin C = ascorbic acid
Menaquinone Vitamin K
Lipoate Not required in diet
Coenzyme Q Not required in diet
55

Enzyme classification: EC system

EC X.Y.Z.W X = class
Y = subclass
Z = sub-subclass
W = the serial number of the enzyme in its sub-subclass

n° Class Type of reaction catalyzed

EC 1 Oxidoreductases + + Transfer of e- = oxidation/reduction reactions

Transfer of a functional group (e.g. methyl,

EC 2 Transferases + + glycosyl or phosphate group)

EC 3 Hydrolases + + Hydrolysis of various bonds

Cleavage of various bonds by means other than

EC 4 Lyases + hydrolysis and oxidation

EC 5 Isomerases Isomerization changes within a single molecule

Formation of covalent bonds coupled with the

EC 6 Ligases + + + hydrolysis of a diphosphate bond in NTP
Movement of ions / molecules across membranes
EC 7 Translocases
or their separation within membranes
56

How enzymes work ?

• Create an energetically favorable environment for the reaction to take place

• Active site
E+S ES EP E+P

Transition state (‡)

Free energy, G

ΔG‡S®P uncat
‡ Enzymes lower the activation energy
ΔG‡S®P cat
ES EP
S ΔG’° = biochemical standard free energy
Ground 25°C (298°K)
state P Partial pressure of each gas = 1 atm
Ground Concentration of each solute = 1 M
state pH = 7
Reaction coordinate
[P]
ΔG’° = – RT ln K’eq K’eq =
Enzymes do not change [S]
• K’eq R = gas constant = 8,315 J.mol-1K-1
T = temperature °K
• ΔG’°
• ΔG’
57

Free energy

ΔG’° are additive For redox reactions: standard reduction

potential E’° is used to calculate ΔG’°
A B ΔG’°1 A + 2 e- + 2 H+ AH2 E’°A

B C ΔG’°2 BH2 B + 2 e- + 2 H+ -E’°B

A C ΔG’° = ΔG’°1 + ΔG’°2 A + BH2 AH2 + B ΔE’° = E’°A - E’°B

ΔG’° = – nF ΔE’° n = number of e- transferred

F = Faraday constant = 96500 C

Actual free energy of a reaction changes depends on reactant and product concentrations
[C]c [D]d
aA + bB cC + dD ΔG’ = ΔG’° + RT ln Q Q= = reaction quotient
[A] [B]
a b

ΔG’ Reaction
<0 Proceeds spontaneously forward Exergonic
=0 Is at equilibrium
>0 Cannot occur spontaneously – an input of free energy is required Endergonic
Proceeds spontaneously in reverse
58

Free energy changes over reactions of Glycolysis in erythrocytes

Glycolysis Glucose 5000 µM ΔG’° (kJ.mol-1) ΔG’ (kJ.mol-1)
ATP
ADP -16.7 -34
Glucose-6-P 83 µM
1.67 -2.9
Fructose-6-P 14 µM
ATP
ADP -14.2 -19
Fructose-1,6-bisP 31 µM large negative free
23.9 -0.23 energy changes =
Dihydroxyacetone-P Glyceraldehyde-3-P 140µM 19 µM 7.56 2.4 reactions
NAD+ 6.3 -1.29
NADH,H+ thermodynamically
1,3-bisphosphoglycerate 1 µM irreversible
ADP
ATP -18.9 0.09
3-phosphoglycerate 120 µM
4.4 0.83
No reaction is at
equilibrium
2-phosphoglycerate 30 µM
1.8 1.1
Concentrations are
Phosphoenolpyruvate 23 µM
ADP at steady state
ATP -31.7 -23
Pyruvate 51 µM
TOTAL: TOTAL:
ATP: 1850 µM -73.97 -98.27
ADP: 140 µM
Pi: 1000 µM
59

How enzymes work ?

• Create an energetically favorable environment for the reaction to take place

• Lower the activation energy
• Enzymes do not change
• Reaction equilibrium (K’eq)
• Thermodynamic feasibility of the reaction (ΔG’)
• Enhance reaction rate (velocity)
d[P] Reaction rate is proportional
reaction rate =
dt • Concentration of substrates (reactants)
• Rate constant k

First order reactions Second order reactions Third order reactions

S P (+ Q) S1 + S2 P1 + … S1 + S2 + S3 P1 + …

V = k [S] V = k [S1] [S2] V = k [S1] [S2] [S3]

EC 3: hydrolases EC 1: oxidoreductases EC 1: oxidoreductases

EC 4: lyases EC 2: transferases EC 2: transferases
EC 5: isomerases EC 6: ligases
60

Initial Velocity First order reactions

• the amount of product formed increases with time

• a time is reached when there is no net change in the concentration of S or P
• the enzyme is still actively converting S into P and visa versa, but the reaction equilibrium
has been attained

• the initial velocity (V0) for

each substrate concentration
S P is determined from the slope
of the curve at the beginning
d[P] of a reaction, when the
reaction rate =
dt reverse reaction is
insignificant

= Simplifying approach

Initial velocity represents best the enzyme activity – no factors that can decrease it:
• Inhibition products
• pH changes
• Denaturation of enzyme
61

Steady-state First order reactions

• Pre-Steady state: very short to be observed

• Steady state:
E+S ES EP E+P
• [ES] constant
• V0 reflects the steady state = approximation
62

Michaelis-Menten Kinetics First order reactions

• simple model that accounts for most of the features of enzyme – catalyzed reactions
E+S ES EP E+P

• Postulation of the reversible formation of ES

Leonor Michaelis

[S0]
V0 = Vmax
Maude Menten Km + [S0]
63

Michaelis-Menten Kinetics First order reactions

k1 k2 k1 k2
E+S ES EP E+P E+S ES E+P
k-1 k-2 k-1 k-2
Very
fast

Changes in the Concentration of Reaction Participants = formation rates – breakdown rates

d[S]
Rate of [S] evolution = = -k1 [E] [S] + k-1 [ES]
dt
d[E]
Rate of [E] evolution = = -k1 [E] [S] + k-1 [ES] + k2 [ES] - k-2 [P] [E]
dt
d[ES]
Rate of [ES] evolution = = k1 [E] [S] - k-1 [ES] - k2 [ES] + k-2 [P] [E]
dt
d[P]
Rate of [P] evolution = = k2 [ES] - k-2 [P] [E]
dt
= reaction rate
64

Michaelis-Menten Kinetics First order reactions

k1 k2 k1 k2
E+S ES EP E+P E+S ES E+P
k-1 k-2 k-1 k-2
Very
fast

Changes in the Concentration of Reaction Participants = formation rates – breakdown rates

d[S]
Rate of [S] evolution = = -k1 [E] [S] + k-1 [ES]
dt
d[E]
Rate of [E] evolution = = -k1 [E] [S] + k-1 [ES] + k2 [ES] - k-2 [P] [E]
dt
d[ES]
Rate of [ES] evolution = = k1 [E] [S] - k-1 [ES] - k2 [ES] + k-2 [P] [E]
dt
d[P]
Rate of [P] evolution = = k2 [ES] - k-2 [P] [E] 1st assumption: initial rate
dt
= reaction rate = V0 [P] ≅ 0
k-2 [P] [E] ≅ 0
65

Michaelis-Menten Kinetics First order reactions

k1 k2 k1 k2
E+S ES EP E+P E+S ES E+P
k-1 k-2 k-1 k-2
Very
fast

Changes in the Concentration of Reaction Participants = formation rates – breakdown rates

d[S]
Rate of [S] evolution = = -k1 [E] [S] + k-1 [ES]
dt
d[E]
Rate of [E] evolution = = -k1 [E] [S] + k-1 [ES] + k2 [ES]
dt
d[ES]
Rate of [ES] evolution = = k1 [E] [S] - k-1 [ES] - k2 [ES] = 0
dt
d[P]
Rate of [P] evolution = = k2 [ES] 2nd assumption: steady state
dt
= reaction rate = V0 [ES] ≅ constant
d[ES]
=0
dt
66

Michaelis-Menten Kinetics First order reactions

k1 [E] [S] - k-1 [ES] - k2 [ES] = 0

k1 ([Et] - [ES]) [S0] - k-1 [ES] - k2 [ES] = 0
3rd & 4th assumption
• Total enzyme concentration is constant:
[Et] = [E] + [ES] [E] = [Et] - [ES]
k1 [S0] [Et]
• [S] >>> [E] [S0] = [S] + [ES] [S0] ≅ [S] [ES] =
k1 [S0] + k-1 + k2
k-1 + k2 [S0] [Et]
Km = [ES] =
k1 [S0] + Km

d[P] [S0]
= k2 [ES] = V0 V0 = k2 [Et]
dt [S0] + Km

The maximum velocity occurs when the enzyme is saturated

Vmax = k2 [Et]
[S0]
V0 = Vmax = Michaelis-Menten equation
[S0] + Km
k2 = kcat = turnover number
67

Michaelis-Menten Kinetics First order reactions

• If [S] >> Km : the rate of product formation is

maximum
d[P]
= Vmax = kcat [Et]
dt

• If [S] = Km : the rate of product formation is half of

the maximum
d[P] Vmax kcat [Et]
= =
dt 2 2

[S0] • If [S] << Km : the rate of product formation depends

V0 = Vmax on both [E] ≅ [Et] and [S] through the efficiency
Km + [S0]
constant kcat/Km
d[P] [S] kcat
Vmax = kcat [Et] = Vmax = [Et] [S]
dt Km Km
68

Michaelis-Menten Kinetics First order reactions

[E3]
Reaction velocity

Reaction velocity
[E2]

[E1]

Enzyme concentration Substrate concentration

[E1] < [E2] < [E3]

Vmax increases
Km is constant
69

Michaelis-Menten Kinetics First order reactions

One substrate – one product

• EC 5: isomerases
k1 k2 [S0]
E+S ES EP E+P V0 = [Et] k2
k-1 k-2 k-1 + k2
[S0] +
Very k1
fast
kcat Km

One substrate – 2 products

• EC 3: hydrolases
• EC 4: lyases
k1 k2 k3
E+S ES EPQ EQ + P E+P+Q
k-1 k-2 k-3
Very k2 k3 [S0]
V0 = [Et]
fast k2 + k3 k k (k + k )
[S0] + -1 23 -1 2
k1 (k2 + k3)
kcat Km
70

Michaelis-Menten Kinetics Second order reactions

2 substrates – 2 products A+B P+Q • EC 1: oxidoreductases

• EC 2: transferases
Sequential Mechanisms
k1 k2 k3 k4
E+A+B EA + B EAB EPQ EQ + P E+P+Q
k-1 k-2 k-3 k-4
Very
fast Random: either A or B may bind to the enzyme
first, followed by the other substrate
A B P Q Ex: EC 2.7.4.3 adenylate kinase:
ATP + AMP « 2 ADP
k1 k-1 k2 k-2 k3 k-3 k4 k-4 Ordered: A (the leading substrate) must bind to
the enzyme first before B can be bound
E EA EAB EPQ EQ E Ex: EC 1.1.1.1 alcohol deshydrogenase:
NAD+ + CH3-CH2-OH « NADH,H+ + CH3-CHO

k3 k4 [A] [B]
V0 = [Et]
k3 + k4 k3 k4 k (k + k ) k k (k + k )
[A] [B] + [B] + [A] 4 -2 3 + -1 4 -2 3
k1 (k3 + k4) k2 (k3 + k4) k1 k2 (k3 + k4)
kcat KmA KmB
71

Michaelis-Menten Kinetics Second order reactions

2 substrates – 2 products A+B P+Q • EC 1: oxidoreductases

• EC 2: transferases
Ping-Pong Mechanisms
k1 k2 k3 k4
E+A+B EA + B FP + B F+P+B FB + P EQ + P E+P+Q
k-1 k-2 k-3 k-4
Very Very
fast fast
Ping: The leading substrate A binds to E, change
A P B Q it to a chemically different form F and the first
product P is released
k1 k-1 k2 k-2 k3 k-3 k4 k-4
Pong: The second substrate B can be bound by F,
the second product Q is formed and the enzyme
E EA FP F FB EQ E is regenerated to the original form E
Ex. EC 2.6.1.x transaminases
amino acid1 + keto acid2 «
k2 k4 [A] [B] keto acid1 + amino acid2
V0 = [Et]
k2 + k4 k (k + k ) k (k + k )
[A] [B] + [B] 4 -1 2 + [A] 2 -3 4
k1 (k2 + k4) k3 (k2 + k4)
kcat KmA KmB
72

Michaelis-Menten Kinetics

2 substrates – 2 products A+B P+Q

Ping-Pong Mechanisms

EC 2.6.1.1
Glutamate aspartate
aminotransferase

L-glutamate + oxaloacetate «
2-oxoglutarate + L-aspartate
73

Michaelis-Menten Kinetics Third order reactions

3 substrates – n products A+B+C P+Q+… • EC 1: oxidoreductases

• EC 2: transferases
Many possibilities & combinations: • EC 6: ligases
• Sequential Mechanisms
• Random
• Ordered
• Ping-Pong Mechanisms

Ex. ordered ter–ter mechanism: EC 6.2.1.4 succinyl-CoA synthetase

succinyl-CoA + GDP + Pi « succinate + GTP + CoA-SH

B A C Q R P

GDP succinyl-CoA Pi CoA-SH succinate GTP

A B C P Q R
k1 k-1 k2 k-2 k3 k-3 k4 k-4 k5 k-5 k6 k-6

E EA EAB EABC EPQR EQR ER E

74

Michaelis-Menten Kinetics Third order reactions

3 substrates – n products A+B+C P+Q+… • EC 1: oxidoreductases

• EC 2: transferases
Many possibilities & combinations: • EC 6: ligases
• Sequential Mechanisms
• Random
• Ordered
• Ping-Pong Mechanisms

Ex. Ping-pong mechanism: EC 1.2.1.12 glyceraldehyde-3-P dehydrogenase

glyceraldehyde-3-P + NAD+ + Pi « 1,3-diphosphoglycerate + NADH,H+

B A C Q P

glyceraldehyde-3-P NADH,H+ NAD+ Pi 1,3-diphosphoglycerate

B P A C Q
k1 k-1 k2 k-2 k3 k-3 k4 k-4 k5 k-5

EA EAB FPB FB FAB FABC EAQ EA

75

Effect of Various Types of Inhibitors

Inhibition Type Rate Equation Apparent Km Apparent Vmax
None V = Vmax [S]/([S] + Km) Km Vmax
Competitive V = Vmax [S]/([S] + Km (1 + [I]/Ki)) Km (1 + [I]/Ki) Vmax
Noncompetitive V = Vmax [S]/((1 + [I]/Ki) ([S] + Km)) Km Vmax /(1 + [I]/Ki)
Uncompetitive V = Vmax [S]/(Km + [S] (1 + [I]/Ki’)) Km /(1 + [I]/Ki’) Vmax /(1 + [I]/Ki’)
Mixed V = Vmax [S]/((1 + [I]/Ki) Km + (1 + [I] Ki’ [S]) Km (1 + [I]/Ki)/(1 + [I]/Ki’) Vmax /(1 + [I]/Ki’)

k1 k2

1/V
none
E+S ES E+P Competitive
k-1 k-2 Noncompetitive
Uncompetitive
k3
[E] [I] Mixed
E+I EI Ki =
k-3 [EI]

k3 [ES] [I]
ES + I EIS Ki’ =
k-3 [EIS]

1 / [S]
76

Non Michaelis-Menten Kinetics enzymes

• Enzymes that regulate the metabolism = regulatory enzymes

• Display increased or decreased activity in response to certain signals
• At key point on the metabolic map
• At the start of metabolic pathways
• At junctions between metabolic pathways
Covalently modulated enzymes
• Control rates
• Reversible & covalent
• Channel metabolites
modification by other enzymes
• Phosphorylation
Allosteric enzymes • Methylation
• Reversible & Non-covalent binding of regulatory • Adenylation
compounds • Ribosylation
• Homotropic: • …
• modulator = substrate • Irreversible & covalent
• Heterotropic: modification by other enzymes
• modulator ≠ substrate • Proteolysis
• = allosteric modulator
• binds at sites different than the active site Other mechanisms
• Conformational changes • Interaction with regulatory
• Large proteins proteins
• Multimeric protein (often) • Degradation
77

Allosteric enzymes

Cooperative behavior = binding of a certain ligand influences the affinity of the protein to a
further ligand of the same (homotropic) or another type (heterotropic)

• Positive • Negative

E+S ES E+S ES

Reaction velocity
ES + S Fast ES2 ES + S Slow ES2
Faster Slower
ES2 + S ES3 ES2 + S ES3
… …
- positive cooperativity
- no cooperativity =
Michaelis-Menten
[ESn]
The binding constant KB = - negative cooperativity
[E] [S]n

KB [S]n
V0 = Vmax n = Hill coefficient Substrate concentration
1 + KB [S]n
78

Non Michaelis-Menten Kinetics enzymes

79

Kinetic parameters of enzymes

• ACE, acetylcholine esterase

• CAN, carbonic anhydrase
• CCP, cytochrome c peroxidase
• FUM, fumarase
• Rubisco, ribulose-1,5-bisphosphate
carboxylase oxygenase
• SOD, superoxide dismutase
• TIM, triosephosphate isomerase

• enzymes operating in secondary metabolism are, on average, ∼30-fold slower than those
of central metabolism
• Substrate low molecular mass and hydrophobicity appear to limit KM optimization
Bar-Even et al. Biochemistry (2011) 50, 4402-4410
80

Bibliography
Nelson & Cox. Lehninger Principles of Biochemistry. Frey & Hegeman.
New York: W H Freeman & Co Enzymatic Reaction Mechanisms.
3rd Ed. (2000) - 4th Ed. (2005) - 5th Ed. (2008) - 6th Ed. (2012) New York: Oxford University Press (2007)

Berg, Tymoczko & Stryer. Biochemistry. Smolke.

New York: W H Freeman & Co The Metabolic Pathway Engineering Handbook
5th Ed. (2002) - 6th Ed. (2006) - 7th Ed. (2010) - 8th Ed. (2015) Fundamentals - Tools and Applications
CRC Press
1st Ed. (2009)

Garrett & Grisham. Biochemistry. Stephanopoulos, Aristidou & Nielsen.

Belmont, CA: Thomson Brooks/Cole Metabolic Engineering: Principles and
3rd Ed. (2004) - 4th Ed. (2010) - 5th Ed. (2012) - 6th Ed. (2016) Methodologies
Academic Press - 1st Ed. (1998)