Metabolic Biochemistry Notes (all lectures) PDF

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METABOLIC BIOCHEMISTRY (91320)Metabolism  complex of physical and chemical processes occurring within a living cell or organism that are necessary for the maintenance of lifeLife  study of the chemical substances and vital processes occurring in living organisms o E = 1 cell > 10 3 chemical...

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Laura Mooney 13251006

METABOLIC BIOCHEMISTRY (91320) Metabolism  complex of physical and chemical processes occurring within a living cell or organism that are necessary for the maintenance of life Life  study of the chemical substances and vital processes occurring in living organisms o E.coli = 1 cell > 103 chemical reactions vs. Homosapiens > 1014 cells = > 1017 chemical reactions Subject Overview (1) Biochemical properties of enzymes and the regulation of enzyme activity (2) Bioenergetics, the role of energy in determining metabolic pathways and the major energy-donating compounds used in metabolic reactions (3) Major metabolic pathways regulating synthesis and breakdown of carbohydrates, lipids, proteins, amino acids and nucleotides (4) The mechanisms that compartmentalise metabolic activity and integrate fuel metabolism (5) Metabolic adaptations that result from different dietary regimens (e.g. starvation, diabetes, and endurance sport) (6) Genetic defects that disrupt pathways to cause metabolic disease (7) Basic experimental techniques used in enzyme analysis

Bioergenetics Categories of metabolism Glycolysis  breakdown of glucose by enzymes, releasing energy and pyruvic acid Catabolism = breakdown to yield energy e.g. glycolysis, ATP o Uses stored energy and releases as free energy Anabolism = construct into a more complex energy, requires energy e.g. peptide (short chains of AA) o Energy stored in chemical bonds between AA o Synthesis of compounds needed by the cell e.g. carbohydrates, lipids, proteins Metabolites  substrates and intermediates involved in metabolism Sources of energy (1) Carbohydrates  in food as starch, sugar or cellulose, yield glucose upon digestion  ATP, simple (monomer glucose/fructose or dimer sucrose) or complex (storage: starch, cellulose, glycogen) (2) Proteins  building blocks of cells (e.g. haemoglobin, antibodies, actin), yields AA and nitrogen Primary = AA sequence Secondary = regular sub-structures e.g. alpha helix or beta sheet Tertiary = 3D structure Quaternary = arrangement of tertiary structures (3) Fats and lipids  highly concentrated e.g. energy reserves, cell membrane components, insulation, yield fatty acids (carboxyl group, hydrocarbon chain) and acetyl CoA upon digestion, 1

Laura Mooney 13251006 saturated (no-double bonds) or unsaturated (presence of double bond = kinks), triglyceride has 3 fatty acid chains (phosphatidyl choline is a common lipid in cell membranes) Adenosine Triphosphate o Energy stored in covalent phosphate bonds

ATP = carrier of chemical energy NAD+ = carrier of H and electrons (more reduced = most energy, accepted the most electrons)

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o o o

Laura Mooney 13251006 Systematic study of the unique chemical fingerprints that specific cellular processes leave behind Metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organisms Metabolic profiling can give an instantaneous snapshot of the physiology of the cell

Fundamental laws of thermodynamics Thermodynamics = brank of physics that is concerned with energy transfer o Principles describe flow and exchange of heat, energy and matter within a system o Metabolic pathways are based on the utilisation and transformation of energy First law of thermodynamics – Conservation of energy o Able to transform energy, but it cannot be created or destroyed e.g. light  chemical, electrical  thermal o Energy before = Energy after o Chemical energy to useable energy Second law of thermodynamics – Entropy o The universe tends towards increasing disorder i.e. entropy (energy lost as heat) o In all-natural processes, the entropy of the universe increases o The loss of energy to the surrounding environment results in an increase of disorder or entropy

Preferred energy source o Loss of reduced carbons, lots of free energy  fatty acid (37kj per gram of energy) o Less reduced carbons, less free energy (16kj per gram of energy) o Carbohydrates are used FIRST (more easily oxidised)  citric acid cycle  ATP o Fatty acids are hydrophobic, need a lot of energy and O2 to catabolised (used for LT storage, does not need H2O) o Glucose is polar and broken down more quickly

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Energy changes in a chemical reaction

Gibbs Free energy (G)

o o o

Energy that can be harnessed to do work Energy goes into making macromolecules and energy goes out when these macromolecules are broken down Determine if favourable and/or spontaneous

Positive △G = unfavourable reaction (energy required to drive the reaction) e.g. glucose monomers  glycogen (low entropy) Negative △G = favourable reaction (reactants have more energy than products) = energy released, favourable e.g. highly ordered glycogen  glucose (high energy)

△G (spontaneous) = △H (enthalpy) – T△S (entropy) (T is temperature is Kelvin)

Enthalpy (H)

Entropy (S)

o o

State of disorder Quantitative value for the level of randomness or disorder in a system

Positive △S = increasing disorder, spontaneous, favourable Negative △S = increasing order (lack of entropy), organised, unfavourable reaction 4

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o

Thermodynamic restraints Difficult to measure change in entropy (S)  need to integrate Keq to determine change in free energy

o

Ratio of products to reactants at equilibrium  indicates direction of reaction

o

Favourable  more energy on the side of products

Quantifying thermodynamic restraints

o

Change in free energy in a standard state △GO standard state (kJ/mol) = – R (J/mol) . T ln K+eq

o

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If △GO > 0, products have more energy than reactants = unspontaneous/unfavourable

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Coupling reactions o o

Drives unfavourable reactions (positive change in free energy) forwards Usually by adenosine triphosphate (high energy bonds between them)  recyclable

Metabolic coupling reactions (REDOX) o Oxidation-reduction reactions occur together (coupled) o Oxidising agent is reduced o Key role in the flow of energy in biological reactions o Electrons carry energy with them from one molecule to another o Important for cellular respiration and photosynthesis

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o o o

Key role in flow of energy in biological reactions Electrons carry energy with them from one molecule to another Energy production during cellular respiration and photosynthesis are entirely based on oxidation-reduction reactions

Electron carriers NAD+ Nicotinamide Adenine Dinucleotide o Carrier of H+ and electrons o Derived from vitamin B3 (niacin) o Involved with enzyme reactions  coenzyme

NAD deficiency Either a deficiency in niacin (vitamin B3) or a poor ability to use niacin, a precursor for an Cause important cofactor in energy metabolism (NAD) Symptoms Diarrhoea, dermatitis, dementia Treatment Niacin supplementation Activation energy o 7

Defines the rate of reaction

Laura Mooney 13251006 o

Activation energy is always positive, free energy is required

High energy required = slow reaction Low energy required = fast reaction Initial input of energy required to bring reactants into a position that allows them to react with one another to make the products Introduction to enzymes Changing the activation energy o Enzymes are highly specific and recyclable o Catalyse reactions  increase rate of reaction without themselves being changed o All enzymes are proteins, but not all proteins are enzymes Enzyme catalysis o No change in free energy o Increasing likelihood that reaction with occur by lowering activation energy

Enzyme specificity o Size & shape (lock/key), electrostatic repulsion and attraction, hydrophobicity/hydrophilicity o Shape of the enzyme remains unchanged o Enzymes are eventually degraded and produced as needed to maintain homeostasis Enzyme nomenclature and classes o No 6 – components stay the same, just rearranged

Negative = exergonic (energy released)

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Keq = products / reactants

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Enzyme Kinetics Enzyme-substrate interactions o o o

Enzymes affect reaction rate Lock & key = substrate must have a perfect fit, otherwise there is no binding (OUTDATED) Induced fit = enzyme will slightly alter its active site to fit a substrate (ACCEPTED) (will not be permanently changed)

Reaction Rate o o

Measure of the amount of reactant converted into product per unit time Determined by concentration of chemicals and rate constant (k)

Equilibrium constants (K) o Forward and reverse reactions occur simultaneously o No net change  plateau

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Steady state kinetics o

State of reaction when concentration of enzyme substrate complex is constant i.e. always a constant amount of substrate and enzyme

Michaelis-Menton and Lineweaver-Burk plot Michaelis-Menton plot o Enzyme-catalysed reactions are saturable (enzyme concentration is constant throughout reaction) o Amount of substrate is gradually increased o Determine reaction rate/velocity over time  plateau/Vmax Km = half of the enzyme are bound to substrate

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High vs. low Km High Km = slow reaction rate, longer time to reach Vmax Low Km = lower substrate concentration for enzyme to have half sites bound, higher rate

Limitations of Michaelis-Menton

o o

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y=mx+b

o Difficult to determine on the hyperbola, points and Vmax are estimated Solution: Lineweaver-Burke double reciprocal plot (rearrange above equation)

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Enzyme Regulation Reversible and irreversible inhibitors Reversible inhibitors o Rapid dissociation of inhibitor from enzymes  used mainly in metabolism Competitive inhibitors Vmax unchanged Km increased

o o o

Dock at enzyme active site Substrate cannot bind, and product cannot be formed Resembles substrate

More substrate added  more diluted inhibitor  eventually reach Vmax  slower

Inhibitor binds to ACTIVE SITE

Uncompetitive inhibitors

o o o o

FLEETING  product cannot be formed Dock at another site on the enzyme (allosteric site) Only binds when enzyme and substrate are docked (enzyme substrate complex) Add more substrate to swamp out inhibitor and ultimately reach SAME Vmax

o o o

Inhibitors can bind free enzyme or enzyme substrate complex Product cannot be formed Unable to outcompete non-competitive inhibitors

Vmax decreased Km decreased Inhibitor binds to ALLOSTERIC SITE

Mixed non-competitive inhibitors Vmax decreased Km unchanged Inh A

peting inhibitors – MIXED me by increasing [substrate]  es not reach Vmax

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Irreversible inhibitors o Dissociate very slowly from target enzyme due to tight (covalent) binding o Drugs and toxins  aspirin/cyclooxygenase, penicillin/transpeptidase e.g. Penicillin

Feedback control Regulatory enzymes o Enzyme inhibition can be used in metabolic regulation o Regulatory enzymes respond to inhibition  Regulation by allosteric modulation  Regulation by cleavage  Regulation by covalent modifications 2 types of regulation = positive (more) or negative (less) o 1st enzyme in reaction will be targeted for regulation (a) Regulation by allosteric modulation o Binding of enzyme to NON-ACTIVE (allosteric) site which affects binding of substrate to ACTIVE SITE by altering shape/size o Prevents build-up of intermediates o Allosteric enzymes often work at the first step and at branch points o Allosteric enzymes are often multimeric  group (complex) of subunits (single enzymes) o Binding to one subunit alters overall shape

Allosteric enzymes – deviant behaviour o Do not behave in the classic Michaelis-Menten way o Often sigmoidal shaped i.e. small change in [substrate] leads to a massive change in enzyme activity (b) Regulation by protein cleavage o Zymogens (proenzymes)  enzymes that need to be cleaved (shortened) in order to be in active form e.g. trypsinogen from pancreases (zymogen) cleaved into trypsin (enzyme, active form in gut) o Allows rapid activation

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(c) Regulation by covalent modification

o o o o o o o

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Michaelis-Menten equation mathematically describes the effect of [substrate] on an enzyme catalysed-reaction Lineweaver-Burke plot is a double reciprocal plot that will yield a linear graph Km is the substrate concentration at half its maximum value (1/2 Vmax) High Km indicates an enzyme with low affinity for a substrate Competitive enzyme inhibitors cannot be overcome by increasing substrate concentration, non-competitive ones cannot Competitive inhibitors do not alter Vmax, but increases Km Non-competitive inhibition does not alter Km, but does reduce Vmax

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Glycolysis – CARBOHYDRATES Steps involved and the two phases Thermic effect of food = energy required to break down molecules (complex macronutrients  simple micronutrients) and subsequent energy provided Hardest to break down = protein  carbohydrates  fats

o

Carbohydrate metabolism o Complex carbohydrates  simple carbohydrates  pyruvate (glycolysis)  energy (citric acid cycle) o Breaking down macronutrients into monomers i.e. glucose o Used for ST storage (water soluble, easy to transport, o Fat is preferred for LT storage (water insoluble, easy to compact for storage) o Individual pathways end in citric acid cycle  energy metabolism Oxidation of glucose o Release of energy (spontaneous, - △G when carbon bonds are broken) o Irreversible process

Steps of glycolysis o

o

Need to use 2 molecules of ATP in first phase Energy only given in “payoff phase”

Uses of pyruvate 15

Laura Mooney 13251006 WITH OXYGEN Complete oxidation of glucose = breaks all 6 carbons through acetyl CoA  citric acid cycle (30 ATP) WITHOUT OXYGEN Uses lactic acid, do not break carbon  energy from NADH

o

Ethanol = 2 carbons More carbons broken = more energy

NADH is the reduced electron carrier that carries energy (less than ATP)

Classification of enzymes ‘ase’

Phosphorylation  adds phosphate group, prevents glucose from leaving the cell Glucose  simple 6 carbon monomer, stored as glycogen mainly in liver Start payoff phase with 3 carbons ONLY (double everything else)

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o o o o o

Hydrogen removed and transferred to NAD+ Transfer energy to create NADH Create inorganic phosphates which hold energy By the end of step 7, the 2 molecules of ATP are gained back In step 10, phosphate group is broken by kinase  producing 2 ATP molecules

Total energy input and output Preparatory phase = 2 ATP in (step 1 & 2) Payoff phase = 4 ATP out (step 7 & 10), 2 NADH out (reduced electron carriers) (step 6) Overall = +2 ATP produced (out) If there is a mutation within glycolysis that completely stops the glycolysis pathway, will the cells still be able to generate ATP and energy? o Body will use existing ATP to break down lipids (into fatty acids  citric acid) and proteins (into AA  citric acid) o Keto diet skips glycolysis of carbohydrates

Gluconeogenesis o o 17

Relates to keto diet  generation of glucose from non-carbohydrate sources Mainly occurs in the liver

o o

Laura Mooney 13251006 Main energy source of brain is glucose (stored glycogen in liver is not enough to feed body & brain) Residual pyruvate goes through reverse glycolysis to make glucose

Three bypass reactions to conserve energy Bypass 1: Pyruvate Kinase

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Bypass 2: Phosphofructokinase

Bypass 3: Hexokinase

o o o

GTP also transports energy  generally transports energy, sometimes preferred by substrates To convert 1 pyruvate, using 2 ATP molecules More NADH outside mitochondria = more ADP produced within cell

Total energy and regulation o Due to the net energy deficit if both pathways ran at the same time, only one can occur at a time o Regulated by turning enzymes on and off through allosteric modifications or transcription

If glycolysis stops and there is a mutation within the phosphohexose isomerase enzyme in the glycolysis pathway, will the body still be able to re-generate glucose for the brain (brain still has normal glycolysis and gluconeogenesis function)? o Yes FIRST then no o If body needs, it will convert glucose  glucose-6-phosphate Yes – glycogen enters the system as glucose-6-phosphate No – glycogen from liver then runs out 19

Laura Mooney 13251006 Regulation o o

Once liver detects an increase in glucose from carbohydrates, glucokinase works to STORE excess (continually works) Hexokinase I = high affinity to substrates  binds easily to glucose molecule

Isomers and kinetics

Hexokinase allosteric regulation

Glucose 6-phosphatase abundance o PRIMARLY FOUND IN LIVER (when glucose to converted to glucose 6-phosphate)  responsible for blood glucose regulation o Only found in a few muscles

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Regulation by transcription High blood glucose concentration o Hexokinase gene active o Increase hexokinase production o Activate glycolysis

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Low blood glucose concentration o G6Pase gene active o Increase G6Pase production o Activate glycogenesis o Maintain blood glucose levels

Laura Mooney 13251006 Fructose-2,6biphosphate regulation

Pyruvate kinase & carboxylase regulation

Nucleotide metabolism

Nuclotide metabolism  citric acid cycle

Pentose phosphate pathway

Uses glucose-6-phosphate as a substrate  becomes a pentose phosphate Alternative pathway to make nucleotides (DNA & RNA) NADPH – reducing agent  anti-oxidant, neutralises oxidative molecules Also used to synthesis fatty acids help creates cell membranes Used in cell proliferation and growth i.e. DNA/RNA backbone o 22

Glucose 6-phosphate is oxidised and donates electrons to NADP+ resulting in a pentose sugar

Laura Mooney 13251006 Uses of NADPH and pentose sugars o o o

Reduce reactive oxygen species, decreasing their toxicity (NADPH) Synthesis of fatty acids (NADPH) Cell proliferation and growth (both)

Of the following diets, is glycolysis or gluconeogenesis favoured? Why? Normal (even ratio of carbohydrates, protein and fats) = glycolysis (carbohydrates available) High carbohydrate = glycolysis (carbohydrates available) High protein = gluconeogenesis (no carbohydrates available  body makes own glucose) Starvation = glycolysis then gluconeogenesis Normal diet for 3 days then high protein = glycolysis

Pyruvate Dehydrogenase o o o o

Need energy to make energy Needs most (protein)  carbohydrates  fatty acids Thermic effect = energy required to break complex macronutrients in to simple macronutrients Prefer to store fatty acids and lipids due to structure

Cytoplasm = anaerobic (glycolysis) Mitochondria = aerobic (pyruvate dehydrogenase and citric acid cycle) o o

End result of pyruvate dehydrogenase (breaking down pyruvate) is acetyl-CoA NO ATP MOLECULES FROM PYRUVATE DEHYDROGEN...