Lecture 8 Metabolism
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Transcript of Lecture 8 Metabolism
Advanced Bioprocess Engineering
METABOLISM
Dr. Ir. Eirini Velliou
28 February 2014
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Dr. Ir. Eirini Velliou
Lecture Overview → Introduction → Enzymes and cellular metabolism → Basic Metabolic Pathways → The Citric Acid Cycle → The Pentose Phosphate Pathway → The Nitrogen Cycle → Metabolism and the environment → Metabolic Regulation → Metabolic Evolution → Energetic of Biological Systems/Redox Reactions → Examples
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Cellular Metabolism → Metabolism (in Greek: µεταβολή/µεταβολισµός= change) → A series of chemical processes/reactions essential for function and maintenance of cells. → Cellular growth → Cellular proliferation → Cellular environmental response → Maintenance/function of intracellular organelles
Anabolism Catabolism © Imperial College London
Cellular Metabolism
→ Catabolism (in Greek: καταβολισµός= change down ) → A series of chemical processes/reactions which lead to the biosynthesis of simple molecules from complex compounds. molecules. → Energy producing process.
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Glucose C02 + H20 +ENERGY
Cellular Metabolism
→ Anabolism (in Greek: αvαβολισµός= change up ) → A series of chemical processes/reactions which lead to the biosynthesis of complex components (glucose…) from simple molecules. → Energy demanding process.
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Cellular Metabolism → General Classification of Metabolic Reactions → Reactions of Energy Production Decomposition of nutrients (glucose) → Biosynthesis of micro-molecules Amino acids, nucleotides → Biosynthesis of macro-molecules Proteins, polysaccharides, DNA, RNA, lipids
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Cellular Metabolism → General Classification of Metabolic Reactions → Reactions of Energy Production Decomposition of nutrients (glucose) → Biosynthesis of micro-molecules Amino acids, nucleotides → Biosynthesis of macro-molecules Proteins, polysaccharides, DNA, RNA, lipids
Enzymes Crucial catalysts for Metabolic Reactions
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Enzymes → Enzymes: 3-D components of protein or (in some cases) RNA nature. → Basic functions/properties of enzymes →Lowering the activation energy that is required for bio-reactions to take place →As bio-catalysts they are not changed nor consumed during a reaction → Co-factors, Co-enzymes
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Enzymes
→ 3-D structure of human glyoxalase
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Source: www.wikipedia.org
Enzymes → Cellular processes: Enzymes participate in the following crucial cellular processes: → Transportation of nutrients to the cell → Intracellular bio-reactions (conversion of nutrients/substrates to cellular components and/or metabolic products) → Excretion of metabolites out of the cell
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S P + X
Product (P)
Intracellular space
Substrate (S)
Enzymes
→ Enzymes - Substrates = Key - Lock binding
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Dr. Ir. Eirini Velliou
Enzymes
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Enzymes → Enzymes-environment: Enzymatic stability is highly affected by environmental factors. TEMPERATURE pH Regulatory molecules
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Enzymes → Enzymes-environment: TEMPERATURE → Enzymatic activity may increase when the environmental temperature is close to the optimal temperature. → Enzymatic activity may decrease when the temperature is much higher: decomposition of the 3D structure leads to enzymatic denaturation.
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Enzymes → Enzymes-environment: pH → pH range 6-8 for most enzymes → Low pH breaks the chemical bonds, therefore, breaking down the 3D enzymatic structure.
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Enzymes → Enzymes-environment: regulatory molecules → Activators: Increase enzymatic activity. → Inhibitors: Decrease enzymatic activity.
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Enzymes →Biochemical/metabolic pathways → Series of reactions → Product of previous is the substrate for the next reaction → Enzymes function synergistically, catalysing the reactions of the pathway
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The ATP/ADP Cycle → Bioenergy: → Essential for: →Transportation of components →Biosynthesis →Growth/Maintenance →Movement → Energy in bio-systems is transferred and stored in the form of ATP molecules.
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ATP + H2O ADP + Pi ΔGo=-7.3 kcal/mol ADP + H2O AMP + Pi ΔGo=-7.3 kcal/mol
The ATP/ADP Cycle → Bioenergy: → Essential for: →Transportation of components →Biosynthesis →Growth/Maintenance →Movement → Energy in bio-systems is transferred and stored in the form of ATP molecules.
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ATP + H2O ADP + Pi ΔGo=-7.3 kcal/mol ADP + H2O AMP + Pi ΔGo=-7.3 kcal/mol
The ATP/ADP Cycle
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Basic Metabolic Pathways
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→ Three basic stages: → 1. Breaking down of macromolecules to simple sub-units. → 2. Breaking down of simple sub-units to acetyl CoA, & limited ATP + NADH. → 3. Oxidation of acetyl CoA to H2O & higher amounts of ATP + NADH.
Basic Metabolic Pathways
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1 2
3
GLYCOLYSIS
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→ Glycolysis=Prelude to 3d stage of catabolism → Final product =pyruvate acid → Pyruvate acid enters the mitochondria for further oxidation to CO2 and H2O. → Glycolysis produces ATP in presence /absence of Oxygen. → Process = main ATP source → Further reaction of pyruvate use up the reducing power produced in reaction 5 of glycolysis →Regeneration of NAD+, essential for glycolisis
The Citric Acid Cycle
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→ Basic Role of Citric Acid: →Provision of electrons (NADH) in biosynthesis →Provision of carbon molecules (-C-) for composition of amino acids →Production of energy
The Citric Acid Cycle
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→ The citric acid cycle nominates aerobic metabolism and it takes place in the mitochondrial area of the cells → Main function: Oxidation of acetyl groups that enter the cycle in the form of acetyl CoA molecules → The reactions form a cycle because the acetyl group is not oxidized directly, but only after it has been covalently added to a larger molecule, oxaloacetate, which is regenerated at the end of one turn of the cycle
The Citric Acid Cycle
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→ The cycle begins with the reaction between acetyl CoA and oxaloacetate to form the tricarboxylic acid molecule called citric acid (or citrate)
The Citric Acid Cycle
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→ Series of enzymatically catalysed reactions then occurs in which two of the six carbons of citrate are oxidized to CO2, forming another molecule of oxaloacetate to repeat the cycle
The Citric Acid Cycle
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→The energy made available when the C-H and C-C bonds in citrate are oxidized is captured in several ways in the course of the citric acid cycle → At one step in the cycle (succinyl CoA to succinate), a high-energy phosphate linkage is created by a mechanism resembling that described for glycolysis
The Citric Acid Cycle
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→ All of the remaining energy of oxidation that is captured is channelled into the conversion of hydrogen - or hydride ion - carrier molecules to their reduced forms →For each turn of the cycle, three molecules of NAD+ are converted to NADH and one flavin adenine nucleotide (FAD) is converted to FADH2 →The energy that is stored in the readily transferred electrons on these carrier molecules will subsequently be harnessed through the reactions of oxidative phosphorylation Pyruvate + 4NAD + FAD 3CO2 + 4NADH2 + FADH2
GDP + Pi GTP
GTP + ADP GDP + ATP
The role of NADH in Oxidative Catabolism
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→ The third stage of metabolism begins with citric acid cycle and ends with oxidative phosphorylation, both of which occur in aerobic bacteria and the mitochondria of eukaryotic cells → NADH=hydrogen provider to bioreactions/power carrier → It is formed by the addition of a hydrogen nucleus and two electrons (a hydride ion, H-) to nicotinamide adenine dinucleotide (NAD) → Because this addition occurs in a way that leaves the hydride ion held in a high-energy linkage, NADH acts as a convenient source of readily transferable electrons in cells → NAD acts similarly to ATP which is as a convenient source of readily transferable phosphate groups: It acts as a convenient source of transferable electrons
The role of NADH in Oxidative Catabolism
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→ NADH=hydrogen provider to bioreactions/power carrier
Anaerobic metabolism of pyruvate
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→ Pyruvate degradation takes place in the cytoplasm towards the formation of:
Ethanol + C02 (bacteria-yeast)
Lactate (mammalian cells)
The pentose phosphate pathway
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Glucose →
Glycolysis
Dr. Ir. Eirini Velliou
The pentose phosphate pathway
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→ From 6 –C- of glucose, 5 –C- sugars + reducing equivalents are generated → Under certain conditions complete oxidation of glucose to CO2 and H2O can take place → The NADP+/NADPH cofactor pair is used as electron carrier system as opposed to aerobic metabolism which utilises the NAD+/NADH cofactor pair → The primary function of this pathway is the generation of reductive equivalents, in the form of NADPH, for further usage in reductive biosynthesis reactions within the cells.
Dr. Ir. Eirini Velliou
The Nitrogen Cycle
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→ Molecules that are part of this cycle: Amino acids and nucleotides. → Amino acids and nucleotides are crucial cellular molecules, composing 2/3 of the cellular dry weight → Nitrogen is un-reactive as a gas, only few species incorporate it into organic molecules, a process known as nitrogen fixation.
Amino acids Nucleotides
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Nitrogen Cycle: Nucleotides
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→ Consist of a base, a phosphoric anion and a sugar → Essential for composition of DNA & RNA molecules → DNA & RNA are synthesized via complex pathways → Nitrogen in the purine and pyrimidine bases is derived from the amino acids: glycine, glutamine aspartic acid → The ribose and deoxyribose sugars are derived from glucose
Dr. Ir. Eirini Velliou
Nitrogen Cycle: Amino acids
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→ Amino acids take part in the bio-synthesis and those not utilised for that, can be oxidized for generation of bio-energy. → Most of their carbon and hydrogen atoms eventually form CO2 or H2O, whereas their nitrogen atoms are shuttled through various forms and eventually appear as ammonia/urea, which are excreted → Each amino acid is processed differently, and a whole constellation of enzymatic reactions exists for their catabolism
Role of coenzymes
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→ Small molecules essential for enzymatic activities → Carrier molecules of a small chemical group, participating in various reactions in which that group is transferred to other molecules → Coenzymes can be covalently linked to their enzyme or may be less tightly bound to it.
Coenzymes Are Involved in the Transfer of Specific Chemical Groups
Coenzyme Group Transferred ATP phosphate NADH, NADPH hydrogen and electron (hydride ion) Coenzyme A acetyl Biotin carboxyl S-Adenosylmethionine methyl
Dr. Ir. Eirini Velliou
Role of coenzymes
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→ ATP acts as an efficient donor of a phosphate group in a large number of phosphorylation reactions, as terminal phosphate linkage in ATP is easily cleaved, with release of free energy → Acetyl coenzyme A (acetyl CoA), carries an acetyl group linked to CoA through a reactive thioester bond. This acetyl group is transferred to other molecules such as a fatty acid (being in formulation) → NADH carries hydride ion, participating in a variety of reactions → Biotin transfers a carboxyl group, taking part in many biosynthetic reactions
Role of coenzymes
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→ Many coenzymes have to be obtained from specific plants of microorganisms present in nutrient sources as they cannot be synthesized by animals. → Vitamins are often precursors of required coenzymes, therefore they are essential nutritional factors for animals.
Metabolic Regulation
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→ Cell growth: Most intermediate molecules contribute as building blocks towards the formulation of macromolecules (e.g. proteins)
When building blocks (e.g. amino acids) are
available in adequate amounts
Biosynthesis
Production of catalyzing
enzymes
Metabolic Regulation
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→ If two substrates for two distinct pathways are available : Enzymes catalysing the pathway for which higher cellular growth takes place and the enzyme catalysing the 2nd pathway is synthesized only after the exhaustion of the first substrate
E. coli growth in medium containing glucose + lactose
EXAMPLE: Glucose is catabolized
β-galactosidase
Glucose is
consumed
β-galactosidase is produced
Growth takes place on lactose
Dr. Ir. Eirini Velliou
Metabolic Regulation
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→ Enzymes Can Be Switched On and Off by Covalent Modification → Feedback control permits the rates of reaction sequences to be regulated continuously and automatically in response to fluctuations in metabolism → Cells have regulate enzymes when longer-lasting changes in activity, occurring over minutes or hours, are required. This regulation is usually achieved by: → The addition of a phosphate group to a specific serine, threonine, or tyrosine residue in the enzyme → The phosphate comes from ATP, and its transfer is catalyzed by a family of enzymes known as protein kinases
Dr. Ir. Eirini Velliou
Metabolic Regulation
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→ Phosphorylation can alter the shape of an enzyme in such a way as to increase or inhibit its activity → The subsequent removal of the phosphate group, is achieved by a second type of enzyme, called a protein phosphatase → Covalent modification of enzymes adds another dimension to metabolic control → It allows specific reaction pathways to be regulated by extracellular signals that are unrelated to the metabolic intermediates themselves → Examples of extracellular signals: hormones and growth factors
Dr. Ir. Eirini Velliou
Metabolic Evolution
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When life began on earth, there was probably little need for such elaborate metabolic reactions
Cells with relatively simple chemistry could survive and grow on the molecules in their surroundings
But as evolution proceeded, competition for these limited natural resources would have become more intense
Organisms that had developed enzymes to manufacture useful organic molecules more efficiently and in new ways would have had a strong selective advantage
In this way the complement of enzymes possessed by cells is thought to have gradually increased, generating the metabolic pathways of present organisms
Two plausible ways in which a metabolic pathway could arise in evolution are illustrated in the figure on the right
Dr. Ir. Eirini Velliou
Metabolic Regulation
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→ The enzymes that catalyze the fundamental metabolic reactions, while continuing to serve the same essential functions, have undergone progressive modifications as organisms have evolved into divergent forms → The amino acid sequence of the same type of enzyme in different living species provides a valuable indication of the evolutionary relationship between these species
Dr. Ir. Eirini Velliou
Metabolic Regulation
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→ An even richer source of information is in the sequences of nucleotides in DNA → Modern methods of analysis allow these DNA sequences to be determined in large numbers and compared between species → Comparisons of highly conserved sequences, which have a central function and therefore change only slowly during evolution can reveal relationships between organisms that diverged long ago → Rapidly evolving sequences can be used to determine how more closely related species evolved
Dr. Ir. Eirini Velliou
Energetics of Biological Systems
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→ Biologically important energy sources: → chemical → light → electrical (involved in energy conversion and membrane transport) → Turnover time of ATP in organisms is in the order of 1-10 s → To utilise the energy stored in ATP there must be a tight coupling of energy-forming reactions (catabolism) and energy-utilising reactions (anabolism)
Oxidation Number
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ON of elements = 0, e.g. N2, H2, O2 ON of ions = charge; Na+ = +1, Fe+3 = +3 Σ in neutral molecule = 0; H2O = 2(+1) + (-2) = 0 Σ in ion = charge; SO4
2- = -2 In multi C molecules average value: C2H5OH → 2C + 6(+1) + (-2) → C = -2 Balance between oxidised and reduced products
e.g. C6H12O6 → 2C2H5OH + 2CO2 C = 0 C = -2 C = +4
Loss of electrons → Oxidised; Gain of electrons → Reduced
Energy yields of reaction
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ΔGo’ = ΔGo + m ΔG’f(H+) ΔGo’: free energy at 1 atm, 1M, pH 7 m: net number of protons in reaction ΔG’f(H+): free energy of formation of a proton at pH 7 = -9.55 kcal at 25oC ΔGo’>0, non-spontaneous endergonic ΔGo’<0, spontaneous exergonic ΔG’r = ΔGo’ + RT lnK
where K = [C]c[D]d/[A]a[B]b
Redox Reactions
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→ Reduction & oxidation → Reactions yield chemical energy → Some utilise O2, many do not → Real basis is electron transfer
→ Half reaction: Fe2+ → Fe3+ + e- → must be matched by electron acceptor reaction since e- cannot exist alone
∴ ½ O2 + e- → ½ O2-
Σ Fe2+ + ½ O2 → Fe3+ +½ O2-
Redox Potential
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→ Substances vary in their tendency to donate electrons → Measured by reduction potential (Eo), i.e., possibility of reaction
→ Oxid + e- → red → i.e., reduction potential measures the potential of the left hand molecule to accept electrons → Negative potential = unlikely → Available energy:ΔGo’ = -n F ΔE’o
→ n – no of electrons (total no per mol) transferred, e.g. glucose to CO2: O = +4 6C/mole = 24 →F – constant (23.06 kcal) →ΔE’o = Eo’ electron accepting pair - Eo’ electron donor pair
Redox Potential-Examples
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→ CO2/glucose = -0.43v → CO2 reduction to glucose is unlikely. More likely for glucose to lose electrons and be oxidised to CO2 → ½ O2/H2O = +0.82v → O2 likely to accept electrons to be reduced to H2O
Electron Carriers
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In most cases transfer of electrons between donor and acceptor is indirect
→ electron carrier
to facilitate cascade of reactions from donor to terminal acceptor is needed:
NAD/NADH = -0.32v
(energy-catabolism)
NADP/NADPH = -0.324v
(biosynthesis – anabolism)
The Importance of Cellular Metabolism in Bioprocessing -Examples-
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Signaling proteins induce thermal resistance to food spoilage bacteria
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Control S1 S2 Control S1 S2
S1: 30-55oC/0.14oC/min
S1 S2
S2: 30-42oC/0.14oC/min
E.coli
Velliou et al. 2012 (Journal of Thermal Biology)
Components in the growth medium may facilitate microbial growth
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Van Derlinden et al. 2008 (Journal of Applied Microbiology)
Presence of molecular chaperone TMAO protects the cells leading to continuation of growth or at least survival
Effect of growth in a glucose rich vs glucose free environment on the microbial thermal inactivation
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Velliou et al. 2011 (Procedia Food Science)
Presence of glucose in the growth medium leads to an increased thermal resistance of E. coli by means of an extension of the lag phase and a reduction of the slope, i.e., slower inactivation, of the inactivation curve at lethal temperatures
E.coli