Lecture 8 Metabolism

58
Advanced Bioprocess Engineering METABOLISM Dr. Ir. Eirini Velliou 28 February 2014 © Imperial College London

description

Metabolism

Transcript of Lecture 8 Metabolism

Page 1: Lecture 8 Metabolism

Advanced Bioprocess Engineering

METABOLISM

Dr. Ir. Eirini Velliou

28 February 2014

© Imperial College London

Page 2: Lecture 8 Metabolism

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

© Imperial College London

Page 3: Lecture 8 Metabolism

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

Page 4: Lecture 8 Metabolism

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.

© Imperial College London

Glucose C02 + H20 +ENERGY

Page 5: Lecture 8 Metabolism

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.

© Imperial College London

Page 6: Lecture 8 Metabolism

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

© Imperial College London

Page 7: Lecture 8 Metabolism

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

© Imperial College London

Page 8: Lecture 8 Metabolism

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

© Imperial College London

Page 9: Lecture 8 Metabolism

Enzymes

→ 3-D structure of human glyoxalase

© Imperial College London

Source: www.wikipedia.org

Page 10: Lecture 8 Metabolism

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

© Imperial College London

S P + X

Product (P)

Intracellular space

Substrate (S)

Page 11: Lecture 8 Metabolism

Enzymes

→ Enzymes - Substrates = Key - Lock binding

© Imperial College London

Page 12: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Enzymes

© Imperial College London

Page 13: Lecture 8 Metabolism

Enzymes → Enzymes-environment: Enzymatic stability is highly affected by environmental factors. TEMPERATURE pH Regulatory molecules

© Imperial College London

Page 14: Lecture 8 Metabolism

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.

© Imperial College London

Page 15: Lecture 8 Metabolism

Enzymes → Enzymes-environment: pH → pH range 6-8 for most enzymes → Low pH breaks the chemical bonds, therefore, breaking down the 3D enzymatic structure.

© Imperial College London

Page 16: Lecture 8 Metabolism

Enzymes → Enzymes-environment: regulatory molecules → Activators: Increase enzymatic activity. → Inhibitors: Decrease enzymatic activity.

© Imperial College London

Page 17: Lecture 8 Metabolism

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

© Imperial College London

Page 18: Lecture 8 Metabolism

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.

© Imperial College London

ATP + H2O ADP + Pi ΔGo=-7.3 kcal/mol ADP + H2O AMP + Pi ΔGo=-7.3 kcal/mol

Page 19: Lecture 8 Metabolism

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.

© Imperial College London

ATP + H2O ADP + Pi ΔGo=-7.3 kcal/mol ADP + H2O AMP + Pi ΔGo=-7.3 kcal/mol

Page 20: Lecture 8 Metabolism

The ATP/ADP Cycle

© Imperial College London

Page 21: Lecture 8 Metabolism

Basic Metabolic Pathways

© Imperial College London

→ 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.

Page 22: Lecture 8 Metabolism

Basic Metabolic Pathways

© Imperial College London

1 2

3

Page 23: Lecture 8 Metabolism

GLYCOLYSIS

© Imperial College London

→ 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

Page 24: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→ Basic Role of Citric Acid: →Provision of electrons (NADH) in biosynthesis →Provision of carbon molecules (-C-) for composition of amino acids →Production of energy

Page 25: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→ 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

Page 26: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→ The cycle begins with the reaction between acetyl CoA and oxaloacetate to form the tricarboxylic acid molecule called citric acid (or citrate)

Page 27: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→ 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

Page 28: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→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

Page 29: Lecture 8 Metabolism

The Citric Acid Cycle

© Imperial College London

→ 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

Page 30: Lecture 8 Metabolism

The role of NADH in Oxidative Catabolism

© Imperial College London

→ 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

Page 31: Lecture 8 Metabolism

The role of NADH in Oxidative Catabolism

© Imperial College London

→ NADH=hydrogen provider to bioreactions/power carrier

Page 32: Lecture 8 Metabolism

Anaerobic metabolism of pyruvate

© Imperial College London

→ Pyruvate degradation takes place in the cytoplasm towards the formation of:

Ethanol + C02 (bacteria-yeast)

Lactate (mammalian cells)

Page 33: Lecture 8 Metabolism

The pentose phosphate pathway

© Imperial College London

Glucose →

Glycolysis

Page 34: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

The pentose phosphate pathway

© Imperial College London

→ 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.

Page 35: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

The Nitrogen Cycle

© Imperial College London

→ 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

Page 36: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Nitrogen Cycle: Nucleotides

© Imperial College London

→ 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

Page 37: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Nitrogen Cycle: Amino acids

© Imperial College London

→ 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

Page 38: Lecture 8 Metabolism

Role of coenzymes

© Imperial College London

→ 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

Page 39: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Role of coenzymes

© Imperial College London

→ 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

Page 40: Lecture 8 Metabolism

Role of coenzymes

© Imperial College London

→ 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.

Page 41: Lecture 8 Metabolism

Metabolic Regulation

© Imperial College London

→ 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

Page 42: Lecture 8 Metabolism

Metabolic Regulation

© Imperial College London

→ 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

Page 43: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Metabolic Regulation

© Imperial College London

→ 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

Page 44: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Metabolic Regulation

© Imperial College London

→ 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

Page 45: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Metabolic Evolution

© Imperial College London

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

Page 46: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Metabolic Regulation

© Imperial College London

→ 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

Page 47: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Metabolic Regulation

© Imperial College London

→ 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

Page 48: Lecture 8 Metabolism

Dr. Ir. Eirini Velliou

Energetics of Biological Systems

© Imperial College London

→ 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)

Page 49: Lecture 8 Metabolism

Oxidation Number

© Imperial College London

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

Page 50: Lecture 8 Metabolism

Energy yields of reaction

© Imperial College London

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

Page 51: Lecture 8 Metabolism

Redox Reactions

© Imperial College London

→ 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-

Page 52: Lecture 8 Metabolism

Redox Potential

© Imperial College London

→ 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

Page 53: Lecture 8 Metabolism

Redox Potential-Examples

© Imperial College London

→ 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

Page 54: Lecture 8 Metabolism

Electron Carriers

© Imperial College London

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)

Page 55: Lecture 8 Metabolism

The Importance of Cellular Metabolism in Bioprocessing -Examples-

© Imperial College London

Page 56: Lecture 8 Metabolism

Signaling proteins induce thermal resistance to food spoilage bacteria

© Imperial College London

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)

Page 57: Lecture 8 Metabolism

Components in the growth medium may facilitate microbial growth

© Imperial College London

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

Page 58: Lecture 8 Metabolism

Effect of growth in a glucose rich vs glucose free environment on the microbial thermal inactivation

© Imperial College London

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