BIOL 2023 After Midterm 2 PDF

Mon Now 18 TCA Kreb is Cycle Citric acid (breathe out (02)...

Mon Now 18 TCA Kreb is Cycle Citric acid (breathe out (02) /Mitochondrial O2 alot !! 0NADA g ->- Erich-rewant - I- ↳ CO2 -CO2 Glycolysis alucose /62) ⑰ e-from i S- : NADHinto W - ~ complex Electron Transport train - reduced " earierin Atoalcolusis e- E -> I picked ↓-8-E gaine (cytoplasm) W E - queenup In up i ⑪ Complex I FADHz IOWCOUC = ATP age 8 I 2x pyruvate (3C) HI - in - ADP Pi + - - I Ht ⑧ Ht 02 PMF proton motor force high com ↳ conc gradient H - 0 intramembrane a matrix Photosynthesis : Wzte+ ATP Sugars = -> Glucose Dark From NADPH to reduce (Om frombine it to photon excites e ron : takes COzt e- usese make glucose higher energy photon amose - to a it level - -i ↑ educa it ~NADP E - 0- = e-carrier I - H20 NADP sabo special Chlorophyl PMF ↓e 0 W ↳ Mn04 PSI PSI Iphotosystem H+ 02 photolusis -> GATP + off plants give protsimesis during - on Chlorophyll a new er getfrom Mn e-back Un gets H2O Chloroplast from  Wed HOU 20 Introduction to Metabolism Niagara Falls Metabolism Addresses two fundamental questions: matter 1. How does a cell extract energy and reducing power from its environment? 2. How does a cell synthesize building blocks of its macromolecules and then the macromolecules themselves? Processes are carried out by a highly integrated network of chemical reactions collectively known as metabolism. Metabolism is complex Over a thousand different chemical reactions occur in an E. coli cell However, there are a limited number of “kinds” of reactions In all life forms, about 100 chemicals play central roles. Central Themes 1. Fuels are degraded, large molecules are constructed step by step in a series of linked reactions called metabolic pathways. 2. An energy currency common to all life forms, ATP (Adenosine triphosphate), links energy yielding pathways to energy consuming pathways 3. The oxidation of electron-rich carbon-based molecules (fuels) powers the formation of ATP 4. Many metabolic pathways, but a limited number of types of reactions and particular intermediates in common.  Living things require constant input of energy 1. mechanical work 2. active transport of molecules in and out of cells 3. synthesis of biomolecules from simple precursors Two major components to metabolism Catabolism rich -e W Fuels (carbohydrates, fats → CO2, H2O + Energy, proteins) simple precursors Anabolism Energy + Simple Precursors → complex molecules oxidized Metabolic pathways have defined start points and end points connected by specific reactions and intermediates Two criteria: 1. individual reactions are specific (provided by the specificity of enzymes) 2. the entire set of reactions must be thermodynamically favourable In metabolism there are many individual reactions that are not thermodynamically favourable so strategies must be employed to drive these reactions forward Free Energy Changes in Chemical Reactions the standard state change in free energy A+B C+D Free Energy (kJ/mol) Free Energy (kJ/mol) ΔGo = negative ΔGo = positive Progression of reaction Progression of reaction ΔGo = negative ΔGo = positive favorable unfavorable spontaneous not spontaneous output of E input of E exergonic endergonic equilibrium favors equilibrium favors right side of equation left side of equation Free Energy The Gibbs Free Energy equation relates the change in Free Energy to changes in Enthalpy and Entropy. Enthalpy = Energy content E↳ Entropy = disorder or randomness ΔG = ΔH -T ΔS When ΔG < 0, the reaction is spontaneous (exergonic reaction) inelin When ΔG > 0, the reaction is not spontaneous (endergonic reaction) - H favours - OG Exothermic vs Exergonic ↳ heat leaves system ↳ involves liberation of heat most times Endothermic vs Endergonic ↳ heat enters system ΔG = ΔH -T ΔS Gasoline is primarily made up of C4-C12 hydrocarbons (ie. butane to dodecane). The C8 alkane Octane is a major component. Notice in the octane example below, heat is given off (therefore ΔH is negative) and disorder increases (so ΔS is positive). Therefore, ΔG will be strongly negative. Note that the sign of ΔG gives no indication of the rate at which a reaction can occur. 2 C8H18 + 25 O2 → 16 CO2 + 18 H2O We must remember that even though a reaction may be spontaneous, it doesn’t mean that the reaction occurs at an appreciable rate. The oxidation (burning) of gasoline is a spontaneous reaction. Lots of heat is liberated so the ΔH is strongly negative The products of the reaction are less ordered than the reactants so ΔS is strongly positive and since ΔG = ΔH -T ΔS ΔG will be negative and the reaction is spontaneous. So why doesn’t this glass of gasoline, which is exposed to oxygen in the air, burst into flames? A glass of gasoline The Standard Free Energy change (ΔGo)is a reference point for comparing chemical reactions under defined (Standard) conditions: 1 atm pressure, 298 K, all reactant and product concentrations starting at 1 M. ΔGo = ΔHo - TΔSo The Biochemical Standard Free Energy change (ΔGo’) also includes: constant pH (pH 7 or [H+] = 10-7 M), constant [H2O] (55 M) The magnitude of ΔGo or ΔGo’ is a measure of how far the standard state is from equilibrium: consider the oxidation of glucose: C6H12O6 + 6 O2 → 6 CO2 + 6 H2O the ΔGo is about -2872 kJ/mol.hand -> right. daoue e Equilibrium for this reaction lies far to the right and standard state initial conditions are far from equilibrium.  every chemical rx will spontaneously proceed until equilibrium has been reached (i.e. where the concentrations of the reactants and products no longer change) The concentrations of the reactants and products at equilibrium can be described by the equilibrium constant Keq, where: for the reaction, A + B C+D nochange , [products] [C][D] reactants conce Keq = [reactants] = [A][B] products there equal -ifthe equilibrium exist in one could determine Keq by setting up a reaction under standard state conditions (e.g. 1 M of A, B, C, and D), letting the rx go to equilibrium, and measuring the [A], [B], [C], [D]. [C][D] > [A][B] where: Keq > 1, then rx is spontaneous as written (left to right) and if (A][B] > [C3[D]Keq < 1, then rx is spontaneous in the reverse direction (right to left) and if K = 1, then both reactants and products exist in equal concentrations (A][B] [CJED] eq = The standard Gibbs change in Free energy (ΔGo) is related to Keq by this equation: ΔGo = -RT lnKeq where, R (gas constant) = 8.314 J/mol K and T is temperature (K) thus Keq can be calculated if ΔG is known, and vice versa: not th modumamically Example -> Favourable the conversion of Glucose-6-phosphate to Fructose-6-phosphate is the 2nd step in Glycolysis ΔGo = -RT lnKeq solve for lnKeq ΔGo = +1.7 kJ/mol reverse rxn is lnKeq = -ΔGo/RT take inverse of log from sides favoured motor Keq = e-ΔGo/RT where e = 2.718 wi+ b is agrees Forevers Mer of - (1700 J mol-1) I productamount = (8.314 J mol-1K-1)(298 K) - 2x reactant Keq = e = 0.504 = [F6P] at equilibrium [G6P] Since Keq < 1, the reverse rx is favoured (twice the amount of reactant vs product) There is a big problem here for life… ΔGo = +1.7 kJ/mol Keq = 0.504 ( i.e Keq < 1) The conversion of G-6-P to F-6-P must occur, it is the 2nd step in glycolysis. Yet it is thermodynamically unfavorable!  Glycolysis * it dnamical e Citric Acid Cycle Citric Acid Cycle Terminal Electron Transport There is a big problem here for life… The conversion of G-6-P to F-6-P must occur, it is the 2nd step in glycolysis. ΔGo = +1.7 kJ/mol Yet it is thermodynamically unfavorable! Keq = 0.504 ( i.e Keq < 1) How do biological systems maintain the reaction directionality required? 1) maintaining out of equilibrium [Reactant]:[Product] ratios 2) coupling an unfavorable rx (+ΔG) to a favorable rx (-ΔG) How do biological systems maintain the reaction directionality required? 1) maintaining out of equilibrium [Reactant]:[Product] ratios The ΔGo value of + 1.7 kJ/mol for G-6-P to F-6-P reaction describes the equilibrium concentrations of reactants to products, but in the cell these concentrations are usually in constant flux. Gibbs introduced this equation to calculate the actual ΔG given the actual concentrations of reactants and products in the cell: [products] [C][D] Δ G = Δ Go + RT lnQ where Q = [reactants] = [A][B] this is the same mass action expression as for Keq Keq is a constant that describes system at equilibrium Q is used to describe system potentially not at equilibrium and reflects actual concentration of products/reactants Relationship between Q, Keq, and ΔG [C][D] A+B C+D Q = [A][B] when Q = Keq actual [A],[B],[C],[D] are at equilibrium concentrations therefore ΔG = 0 and no net reaction when Q < Keq actual [reactant] greater than at equilibrium or actual [product] lower than at equilibrium therefore ΔG < 0 (- ΔG ) fwd reaction favored (formation of products) and rx spontaneous as written when Q > Keq actual [product] greater than at equilibrium therefore ΔG > 0 (+ ΔG ) rev reaction favored (formation of reactants) and rx not spontaneous as written  a went inter ΔGo = +1.7 kJ/mol Keq = 0.504 ( i.e Keq < 1) [reactant] [product] Δ G = Δ Go + RT ln Q -perturbed eq when where Q = actual [products] Manifestation of Le Chatelier’s Principle [reactants]  Δ G = Δ Go + RT ln Q [products] ΔGo = +1.7 kJ/mol where Q = actual [reactants] Keq = 0.504 ( i.e Keq < 1) Keq ratio R:P = 2:1 Q ratio R:P = 6:1 Yactual cone. in cell [G-6-P]actual = 8.3 x 10-5 M [F-6-P]actual = 1.4 x 10-5 M [reactant] [product] Let’s calculate the actual ΔG for rx using actual cellular concentrations of reactant and product Δ G = Δ Go + RT ln Q 372 - -5 Δ G = +1.7 kJ mol-1 + (0.00831 kJ mol-1K-1)(310 K) ln 1.4 x 10-5 M 8.3 x 10 M Δ G = +1.7 kJ mol-1 + (-4.6 kJ mol-1) 20 Δ G = -2.9 kJ mol-1 spontaneously goes backto eq. Question from a previous final exam: ⑧ O in e -K Keg e = S = 0. 1 25 : 2 + 273 , 15=298 15k. O I = I 100 : it 0 Q 05 = = 0. 04 = 040 + RTInQ ⑧ = 5. 7 + (0 008314) (310) (n10 05).. =- 2 How do biological systems maintain the reaction directionality required? 1) maintaining out of equilibrium [Reactant]:[Product] ratios maintaining Q < Keq next 2) coupling an unfavorable rx (+ΔG) to a favorable rx (-ΔG) Coupling an unfavorable rx (+ΔG) to a favorable rx (-ΔG) Consider the reaction: A B ΔG = + 16 kJ/mol this is an unfavorable reaction and will not proceed as written However, if the reaction can be coupled to another reaction, say: C D ΔG = - 30 kJ/mol then the net reaction becomes: A + C B+D and the overall ΔG becomes: ΔG = + 16 kJ/mol ΔG = - 30 kJ/mol ΔG = - 14 kJ/mol Unfavorable reactions (+ΔG) can be made possible by coupling to the hydrolysis of ATP (- ΔG) Transcription Atp used during , used as energy not just The nucleotide ATP ATP + H2O → ADP + Pi (inorganic phosphate) ΔG = -30.5 kJ/mol  Consider reaction: conversion of reactant A to product B A B ΔG = +16 kJ/mol the reaction is not thermodynamically favorable (not spontaneous) but when coupled with the hydrolysis of ATP: A + ATP + H2O B + ADP + Pi ΔG = +16 kJ/mol ΔG = -30.5 kJ/mol free energy changes in coupled reactions are additive Pht bu ΔG = -14.5 kJ/mol AtD Changes - + 4 themodynamically Atp ATP + H2O → ADP + Pi (inorganic phosphate) ΔG = -30.5 kJ/mol Coupling reactions with hydrolysis of ATP can be interpreted very generally: transport of a molecule across a membrane through a transporter protein conformational changes in proteins leading to mechanical work conversion of reactant to product Think about: App mechanisms -certain membrane transporters -helicase function during DNA replication -Rho protein function during termination of transcription and Recall how ATP can participate in a reaction…  1. Amino acid must be activated by adenylation Aminoacyl tRNA (adenine addition to amino acid). Synthetase 2. ATP (adenosine triphosphate) is a reactant. activity 3. Nucleophilic attack on P atom results in formation (Lecture 22) of aminoacyl adenylate intermediate. 4. The 5’ adenine of a tRNA attacks the carbonyl O atom in ester linkage. 5. Amino acid transferred to tRNA and linked via an ester linkage DNA Ligase Activity covalent Okazaki 1 catalysis (Lecture 20) Mg2+ bacterial Active site Okazaki 2 Mg2+ adjacent to Lysine –NH3+ group lowers pKa and promotes deprotonation to -> e-carrier –NH2 4 APP in our alls +OE-- At –NH2 is a stronger nucleophile than –NH3+ Okazaki 1 Okazaki 2 Nicotinamide Adenine Dinucleotide (NAD+) NAD+ e-carrier butalso NAD+ acts as the adenylate donor for ligase enzyme in bacteria + 025- Oh ATP acts as donor for ligase in eukaryotes sources OF AMP which is used to +Oh -- Du  In the preceding ligase and aminoacyl-tRNA transferase examples ATP (or NAD+) donates an adenosine monophosphate to activate a reactant but in other reactions it often performs the same function by donating just a phosphate group instead. Thus, ATP is often called a phosphoryl donor or activated carrier of phosphoryl groups. - comes in to drive unfavourable ↑ , ATP is the universal phosphoryl donor in cells but other molecules can also act as phosphoryl group donors/carriers as well these readily hydrolyze with the release of energy We will see these molecules in subsequent lectures Tendency of ATP to hydrolyze (its phosphoryl transfer potential) is intermediate amongst phosphoryl carriers We can think of this as ATP having an intermediate stability which is one reason why it is the universal energy carrier in all life forms ATP is the principal donor of free energy in all biological systems It is rapidly hydrolyzed in chemical reactions in the cell and must be regenerated from ADP constantly ATP regeneration is a primary purpose behind the oxidation of carbon-based fuels in cells (i.e. catabolism) A significant component of upcoming lectures will concern how ATP is generated Amount of ATP in body = 250 grams Amount of ATP used daily = 60 - 80 kg ↳ Why we needto eat rich food to make lots every few hours Of ATP Redox reactions are the central reactions of life transfer of electrons from a reducing agent (reductant) to an oxidizing agent (oxidant) glucose + O2 → CO2 + H2O Reductants are oxidized with the release of energy which is then used to generate ATP The food molecules we consume (e.g. carbohydrates, fats) are in a reduced state that can be more fully oxidized (ultimately to CO2). The more reduced a fuel molecule, the more energy can be derived from its oxidation methane is not a fuel for humans but it illustrates the point completly oxidized  -> whe we use them forage molecules Fats are more reduced (less oxidized) than carbohydrates They therefore yield more energy upon oxidation This is why fats are a primary energy storage molecule in bodies We will encounter how ATP is generated through the oxidation of reducing agents (fuel molecules) in later lectures. But remember two important aspects of metabolism: Catabolism and Anabolism Catabolism oxidation of reduced fuel molecules to generate ATP which is required to build our own macromolecules. Anabolism essentially building our own reduced macromolecules from more oxidized precursors. So in addition to needing ATP, we need some of the electrons from food molecules to build our own macromolecules. Thus electron carriers are important in the catabolism process (to generate ATP) and the anabolism process (to synthesize our own biomolecules) Also remember, Two main ways to drive forward unfavorable reactions: 1) maintaining out of equilibrium concentrations of reactants/products 2) coupling the reaction to ATP hydrolysis Two main ways to understand the action of ATP 1) thermodynamic perspective 2) mechanistic perspective (i.e. what happens in active site of the enzyme) - Wh + Oh Next class (Friday) we will take up the questions and answers from Midterm 2 Y of exam will be content from after midterm 2 Central Carbon Metabolism I: MOUNOU 25 Glycolysis Henry Hillier Parker. Harvest Time 1 Central Carbon Metabolism A+ NAD8 - B + NADH Purposes: Electrons for energy production (carried by NADH) Acquisition of Carbon and other elements (precursor molecules) Electrons for biosynthetic processes (carried by NADPH) of 3tripped we add trawiese then oxidized Food from back , to molecules them Carbohydrates Fats Proteins Basic scheme consists of: Glycolysis and Fermentation (Today) Citric Acid/Tri-carboxylic Acid (TCA) cycle (Wednesday) Electron Transport Chain (Friday) 2 Proton Motive Force and Oxidative Phosphorylation (Monday)  -> favoured by most cells but not the only one TCA cycle Glycolysis e- Electron Transport Chain 02 e- goto -> which reduces 02- H20 3 Three major Heteroorganotrophic Energy acquisition lifestyles Twe gett can be described in terms of the fate of electrons from organic fuel molecules by consuming molecules rich in es made by the plants animals we eat Aerobic Respiration -> makes ~xns in Cells happen (+8h - - -2) glycolysis, TCA, e- transport and ATP production (oxidative phosphorylation) glucose is the source of electrons O2 is the terminal electron acceptor high amounts of ATP many bacteria, eukaryotes Anaerobic Respiration we can't do this -> glycolysis, TCA, e- transport and ATP production ( oxidative phosphorylation) glucose is the source of electrons non-O2 electron acceptors, e.g. Nitrate, Sulfate high ATP, but less than aerobic respiration many bacteria can carry out both aerobic and anaerobic respiration Fermentation abbreviated system, glycolysis only pyruvate is the terminal electron acceptor low ATP production (substrate level phosphorylation) all cells when necessary, cancer cells some bacteria are obligatory fermenters, yeasts 4 Energy Production in Cells The Flow of Electrons from Food Molecules to waste products Work done grind grain cut wood generate electricity Bob Ross Analogy - water flows spontaneously downhill (dissipation or release of potential energy) 5 -electrons flow spontaneously down a reduction potential pathway (releasing energy)  energy from ↑2- oxidation less energy -adized , -250 kJ/mol glucose -2800 kJ/mol glucose -> mostly convertedto ATD only getsof a sman < fraction the 280015/mol glucose TCA cycle electron transport chain 6 e-rich reduced molecules Glucose-> pyruvate Glycolysis Consumes 2 ATP Produces 4 ATP Net = 2 mol ATP produced per mol glucose 7 + 2 NADH There are 6 Classes of Enzymes Transferase (32.5 %) Oxidoreductase (18.6 %) 1. Oxidoreductase 2. Transferase 3. Hydrolase 4. Lyase 5. Isomerase Ligase (10.9 %) 6. Ligase Hydrolase (22.4 %) Isomerase (5.6 %) Lyase (9.9 %) 8 Oxidoreductase (e.g. alcohol dehydrogenase) oxidation-reduction reactions. 1 or 2 electron transfer reactions - corresponding change in H or O atoms on molecule Transferase (e.g. glucose kinase) transfer molecular groups from donor to acceptor molecules. Groups may be carboxyl, methyl, amino, phosphoryl, carbonyl, acyl groups. ADP 9 Hydrolase (e.g. protease) -> Chymotrypsin cleavage of bonds such as C-O, C-N, O-P accomplished with the addition of water. Lyase (e.g. pyruvate decarboxylase) reactions in which groups (e.g. CO2, H2O, NH3) are removed to form a double bond or are added to a double bond 10 Isomerase (e.g. alanine racemase) catalyze intramolecular rearrangements Ligase (e.g. pyruvate carboxylase) - DNA rep catalyze bond formation between substrate molecules at expense of ATP 11 Reactions in Glycolysis When glucose and other sugars enter the cell, they are phosphorylated. -> phosphate From ATP Reaction 1 Prevents escape out of the cell. Increases reactivity of sugar. Enzymes that transfer a phosphate group are often called kinases. Transferase reaction Reaction 2 =Gulse ~Eise G-6-P is isomerized to F-6-P. This produces a C-1 carbon that is available for phosphorylation. also fructose more amenable to cleavage than glucose. Isomerase reaction 12 Reaction 3 F-6-P is phosphorylated on the C-1 carbon by the enzyme Phosphofructokinase-1. Notice that 2 ATP have been consumed so far. Transferase reaction 13 Reaction 4 Next, the Fructose-1,6-bisphosphate is split into two molecules. Lyase reaction (removal of a group producing double bond and cleavage) Reaction 5 Dihydroxyacetone phosphate is isomerized to G-3-P. Only G-3-P is substrate for subsequent reactions so it is essential that DHAP be converted to G-3-P. Isomerase reaction 14 Glycolysis Consumes 2 ATP Produces 4 ATP Net = 2 mol ATP produced per mol glucose 15 + 2 NADH  AD making Reaction 6 start Glyceraldehyde-3-P is oxidized to Glycerate-1,3-biphosphate, a high energy molecule capable note the phosphate group comes from inorganic P of donating a phosphoryl group. Oxidation-Reduction reaction 16  Reaction 7 Glycerate-1,3-bisphosphate (aka 1,3-bisphosphoglycerate) donates a phosphate to ADP producing ATP. This is called substrate level phosphorylation. & comes from substrate phosphate Transferase reaction coupled reaction 1,3-BPG → G-3-P -49.4 reverse sign ↓ I ADP → ATP +30.5 net -18.9 = thermo- dynamically pavourable note: this is using Standard free energy values 17 Reaction 8 Glycerate-3-P is a poor phosphoryl donor (see table) so it is converted to glycerate-2-P. Isomerase reaction Reaction 9 Glycerate-2-P is converted to Phosphoenolpyruvate, a much stronger phosphoryl donor (see table) 1 () 9) -. I goodat donatiry phosphate strong - th+ Lyase reaction Reaction 10 PEP donates phosphoryl group to ADP forming ATP and pyruvate. (substrate level phosphorylation) Transferase reaction 18 The net products of Glycolysis For every 1 mol Glucose: 2 mol ATP 2 mol NADH 2 mol Pyruvate Pyruvate is still an energy-rich (electron-rich) molecule and will enter into the Citric Acid Cycle where its carbons will be oxidized to CO2 and its electrons will be transferred to NAD+ to form NADH NADH then enters the terminal electron chain where most ATP is produced in process called Oxidative phosphorylation. 19 actual ΔG (kJ/mol) -33.5 Glycolysis -2.5 Consumes 2 ATP -22.5 -84 onewas path - strong weak-th or even tal -1.3 two way path -> -1.7 Produces 4 ATP +1.3 +0.8 -3.3 Net = 2 mol ATP produced -16.7 per mol glucose 20 + 2 NADH Focus: Reaction 1 (hexokinase reaction) Phosphorylation of Glucose 21 Coupling ATP hydrolysis to other reactions Moran pg 330 Why is glucose phosphorylated in the 1st step of glycolysis? 2 reasons mem The phosphorylation of glucose by inorganic phosphate (Pi) is thermodynamically unfavorable. This is why reaction is coupled to hydrolysis of ATP. What metabolic strategy could drive the reaction: glucose + Pi → glucose-6-P, forward? Does this happen? 22 Focus: Reaction 2 (Isomerization of G-6-P to F-6-P) -aldehyde to ketone isomerization Why is glucose -6-P isomerized to fructose-6-P? 2 reasons 23 From Lecture 22 Δ G = Δ Go + RT ln Q [products] ΔGo = +1.7 kJ/mol where Q = actual [reactants] Keq = 0.504 ( i.e Keq < 1) [G-6-P]actual = 8.3 x 10-5 M [F-6-P]actual = 1.4 x 10-5 M Keq ratio R:P = 2:1 [reactant] [product] Q ratio R:P = 6:1 Let’s calculate the actual ΔG for rx using actual cellular concentrations of reactant and product Δ G = Δ Go + RT ln Q -5 Δ G = +1.7 kJ mol-1 + (0.00831 kJ mol-1K-1)(310 K) ln 1.4 x 10-5 M 8.3 x 10 M Δ G = +1.7 kJ mol-1 + (-4.6 kJ mol-1) 24 Δ G = -2.9 kJ mol-1  The Fate of Pyruvate Anaerobic Aerobic Fermentation : pyruvate -> ethanol or Respiration lactate Respiration e.g. yeast, lactic acid bacteria human cells starved of O2 human cells needing extra ATP cancer cells 25  In strictly fermentative organisms only glycolysis is used. This presents a problem because soon all of the NAD+ is reduced to NADH and the flow of electrons (and life) will cease. a -> picksup e aretique ↑ This is why cells that ferment produce organic acids and -> gives backto pyruvate ↓ alcohols as waste products. reconvertt ↳ regenerate t Note that the production of lactate and ethanol requires NADH and produces NAD+, thus restoring cellular pool of NAD+. Lactate or ethanol excreted as waste products carrying the e- 26 with them The Fate of Pyruvate Anaerobic Aerobic Fermentation Respiration Respiration yeast, lactic acid bacteria In fermentation, pyruvate becomes the terminal electron acceptor the net production is 2 ATP (much of the potential energy is excreted as waste products) 27 The Fate of Pyruvate (again) Note that glycolysis etc isn’t just about energy -also Carbon acquisition and macromolecule biosynthesis gluconeogenesis and amino acid fermentation precursor amino acid synthesis precursors TCA cycle 28 Entry of other sugars into glycolytic pathway enters glycolytic pathway ↓ Fructoset glucose 29 Entry of non-carbohydrate fuel molecules into glycolysis 30  Today we examined some features of Glycolysis and Fermentation Next day: the fate of pyruvate as it enters the TCA cycle Glycolysis TCA cycle 31  Wed NOV 27 Metabolism II: Citric Acid Cycle Megascops asio (Eastern Screech Owl) 1  Glycolysis Glucose → Pyruvate 2 ATP 2 NADH 2 Pyruvate In a fermentative organism, than NADH reduces pyruvate to organic alcohol and acid intertwine · waste products, thus regenerating the cellular pool of NAD+. In an organism capable of respiration, pyruvate enters the citric acid cycle and NADH unloads its electrons into the electron transport chain. 2 Glycolysis One 6C Glucose molecule (start) to two 3C pyruvate molecules (end). Glucose is phosphorylated (step 1) from ATP, then isomerized to fructose sugar (step 2) and again phosphorylated (step 3) using ATP to form Fructose 1,6-bisphosphate. This is cleaved to 2 molecules of glyceraldehyde-3 phosphate (steps 4 and 5). In an oxidoreductase reaction, a second phosphate is added (from inorganic phosphate) and electrons reduce NAD+ to NADH (step 6). One of the phosphates on glycerate-1,3, bisphosphate is added to ADP to form ATP (step 7). This ATP synthesis process is called substrate level phosphorylation and can occur because hydrolysis of glycerate- 1,3, bisphosphate has a sufficiently negative ∆G and when coupled with the positive ∆G of forming ATP, generates a reaction that has a net negative ∆G (ie thermodynamically favorable reaction). The resulting glycerate-3-phosphate is re-arranged into phosphoenolpyruvate (steps 8 and 9) which also has a sufficiently negative ∆G of hydrolysis to add its remaining phosphate to ADP to form ATP in a coupled reaction (again, this is called substrate level phosphorylation) (step 10). The remaining molecule is now called pyruvate and would typically enter into the TCA cycle. The NADH formed in step 6 would donate its electrons to the electron transport chain thus regenerating the cellular pool of NAD+. For those organisms that do not have a TCA pathway or electron transport chain, the electrons from NADH are donated to the pyruvate forming either ethanol or lactate as waste products in a process called Fermentation. This step is crucial to regenerating 3 the cellular pool of NAD+. Eukaryotes Glycolysis occurs in the cytoplasm Citric Acid Cycle occurs in mitochondria Prokaryotes Glycolysis and citric acid cycle occurs in cytoplasm The Endosymbiont Theory 4 Mitochondria are obligatory endosymbionts of the eukaryotic cell were once free-living bacteria estimated 2 billion years ago these bacteria became endosymbionts of a larger cell (likely an Archaea), thus producing the eukaryotic cell type that mitochondti Abacteria was ofrom n dained Mitochondrion Gram Negative Bacterial Cell Cytoplasm Cytoplasmic membrane Outer membrane 5 Bacteria Archaea Eukaryote Three domain hypothesis as originally conceived (and what you will see in textbooks) LUCA (Last Universal Common Ancestor) Bacteria Eukaryote Archaea Perhaps a more likely scenario LUCA 6 The TCA Cycle mitochondrion Pyruvate dehydrogenase action links glycolysis to Citric acid cycle ePyruvate is imported into mitochondrion wan linked to coenzyme A by enzyme Pyruvate dehydrogenase. CO2 is liberated. This enzyme can be inhibited by heavy metals (e.g. As, Hg) -> 2 Cleft rate ↳ makes it thermodynamically favourable Oxidation-Reduction reaction 7 ↑We loe Citric Acid cycle earboxylate 3 -> groups e Tri-carboxylic acid cycle -> breathe Krebs cycle -rat - wh i Ec - AND I stage a Stage I 2 G all 3 more e acetate amoxic CO2 oxalo reathed - Gregenerate reduced D - out A Purposes EY Etc oxidized -Adde en -oxidize glucose to CO2 -capture electrons -produce precursors 2 stages out ↑ I breathed + - - 36 -oxidize Carbons -regenerates oxaloacetate 8 The Structure and Function of coenzyme A (coA) -Coenzyme A is a high energy carrier molecule that carries 2 carbon (acetyl) molecules in both catabolic and anabolic processes. At from comes originally - comes from adenosine steine diphosphate structure of Coenzyme A* sometimes written as coASH - ↓ group from byS A 2 carbon molecule attached to Coenzyme A -acetyl coA activates the 2C molecule for addition to oxaloacetate which would be unfavorable otherwise * the biosynthesis of coenzyme A involves ATP, pantothenate (vitamin B5), and cysteine. 9 Life is all about electron transfer Electron transfer is all about Reduction-Oxidation (RedOx) reactions Areduced + Boxidized → Aoxidized + Breduced glucose + 6O2 → 6CO2 + 6H2O reduced oxidized oxidized reduced isocitrate + NAD+ → α-ketoglutarate + CO2 + NADH reduced oxidized oxidized reduced 10 The electron carrier NAD+ (in what role have we encountered NAD+ before?) ↳ ligase active site see ~nitrogen S Nicotinamide Adenine Dinucleotide Hydride ion (H-) 11 DNA Ligase Activity covalent DNA 1 catalysis Mg2+ Active site Mg2+ adjacent to DNA 2 Lysine –NH3+ group lowers pKa and promotes deprotonation to –NH2 I source of ADP which a+ - Ob + –NH2 is a stronger DNA 1 nucleophile than –NH3+ Phosphodiester blu , DNA 2 bond okazali ments Frag - Nicotinamide Adenine Dinucleotide (NAD+) -nicotinamide is a nitrogenous base not used in nucleic acids Adenine and Nicotinamide nucleotides are linked by a phosphoanhydride linkage In bacteria, NAD+ is used to adenylate Lys In eukaryotes and certain viruses, ATP is used to adenylate Lys ATP  In aerobic organisms, the ultimate electron acceptor from reduced fuel molecules is oxygen. Electrons are not transferred directly to O2, but are transferred to special carriers, such as NAD+ (nicotinamide adenine dinucleotide) a reduced molecule is oxidized by NAD+, and NAD+ is reduced to NADH via the transfer of one H atom and 2 electrons During biosynthetic processes, NADPH NAD, where R = H is the carrier of electrons. NADP, where R= phosphate group 14 The electron carrier FAD Look! ATP again. Roles for ATP we have encountered: isoalloxazine -energy currency of cell -ribonucleotide (RNA synthesis) -precursor of dATP (DNA synthesis) -precursor for coA -precursor of NAD+ -precursor of FAD FAD (Flavin Adenine Dinucleotide) 15 The Reactions 16 The TCA Cycle mitochondrion Pyruvate dehydrogenase action links glycolysis to Citric acid cycle Pyruvate is imported into mitochondrion linked to coenzyme A by enzyme Pyruvate dehydrogenase. CO2 is liberated. This enzyme can be inhibited by heavy metals (e.g. As, Hg) 17 Oxidation-Reduction reaction  Reaction 1 1st step in the cycle. Formation of 6-C citrate. Transferase reaction Citrate carrying a tertiary alcohol is Reaction 2 converted to a more reactive aconitase secondary alcohol Isomerase reaction true H X g more reactive 18 Reaction 3 isocitrate dehydrogenase Oxidative decarboxylation of isocitrate to form α-ketoglutarate. The second CO2 and NADH produced. Oxidation-reduction reaction Reaction 4 Oxidative decarboxylation of α-ketoglutarate. Like acetyl-coA, succinyl coA is highly reactive. CO2 and NADH produced. Brac ketoglutarate dehydrogenase from pyruvate 19 Oxidation-reduction reaction Reaction 5 -litte be Cleavage of high energy X X thioester linkage coupled to substrate level succinyl-coA synthetase phosphorylation of ADP. Ligase reaction Reaction 6 Oxidation of succinate to Fumarate using FAD instead of NAD+ A Oxidation-reduction reaction succinate E dehydrogenase note: FAD is Flavin Adenine Dinucleotide which is similar to NAD in that it can accept electrons, but it is not a mobile carrier… 20 it is embedded within the succinate dehydrogenase enzyme complex Reaction 7 Fumarate is hydrated to Malate. fumarase Lyase reaction Reaction 8 Malate is oxidized to oxaloacetate. NAD+ is reduced to NADH. ↳ ETL-AN 7 malate Oxidation-reduction reaction dehydrogenase 21 per glucose 8 NADH 2 FADH2 2 ATP/GTP 6 CO2 1st half cycle oxidation of carbons 2nd half cycle regeneration of oxalacetate 22 Focus: Reaction 8 malate to oxaloacetate reaction is bi cycle going happen -Will ↑ [malate] wan mere than it to some will have go should be then to product taken away (same if product is thingas ↓ above will happen to start cycleagain [malate] [oxaloacetate] in the cell, oxaloacetate is rapidly consumed by the very exergonic unfavorable -> Reaction 1. 23  will be on exam How would you handle this question if it appeared on an exam? When measured under standard conditions, a reaction: ΔGo = -RT lnKeq A+B C+D Δ G = Δ Go + RT lnQ where R (gas constant) = 8.314 J mol-1 K-1 has a ΔG0 = + 2.0 kJ/mol and T (temperature) = 310 K (37oC) a) is this reaction spontaneous (favorable) as written? NO b) calculate the equilibrium constant (Keq) for this reaction and the ratio of reactants/products at equilibrium. * 2 0K5/mol--8 3145mor1K -InKeq. = Req 2 = = e-200%.31/310) = 0. - 46 c) if in the cell, the following concentrations are ambient: [A] = 2 mM, [B] = 10 mM, [C] = 1 mM, [D] = 1 mM a ! (h=200 314(310) = = 0. 05 + 8. (n 10 05). = 57215/mol =- 5. 7K5/mOl What is the actual ΔG for this reaction in the cell? Oh = 2 + 8 314(310) In Q. I need to calculate Q 24  Actual ΔG values for reactions Note that the reactions with actual ∆G values around 0 become very sensitive to out of equilibrium concentrations of either reactants or products which can easily drive the reaction forward as written or, in the reverse direction which is sometimes necessary…. This is called “metabolic flux” 25 The citric acid cycle is an AMPHIBOLIC pathway (both catabolic and anabolic pathways) anabolic processes replenishing reactions 26 Amino acid Biosynthesis from Glycolysis and TCA cycle intermediates (and e- from NADPH) TCA cycle glycolysis NADPH ATP TCA cycle glycolysis TCA cycle 27 glutamate dehydrogenase note the role of NADPH (its not all about energy production, electrons also needed for the reductive biosynthesis of self molecules) glutamine synthetase 28 As a result of Citric Acid Cycle: 6 Carbons in Glucose are oxidized to 6 CO2 and exhaled as waste products Some of the intermediates in the TCA cycle are retained for biosynthetic purposes in the form of chemical precursors for lipids, amino acids, nucleotides, etc. and some of the electrons are retained (by NADPH) for this purpose. Electrons from Glucose are carried to the electron transport chain by NADH (and FADH2) to make lots of ATP (remember, very little ATP has been made during glycolysis and TCA cycle) 29 Final Exam (unofficially): Monday December 16 (9 am -12 noon) Currie Center 30  Fri NOV 29 Metabolism III: Electron Transport and the generation of a Proton Motive Force Ursus maritimus  Glycolysis Citric Acid Cycle ·tion  What he need TCA cycle 3C pyruvate from Glycolysis: carbon 1 oxidized to to know CO2, NAD+ reduced to NADH, and remaining 2 carbons attached to Coenzyme A (CoA). Acetyl-coA is very reactive and the 2 C are added to the 4C oxaloacetate to form 6C citrate (step 1). Carbon 2 of 6C isocitrate is oxidized to CO2 and NAD+ reduced to NADH, forming 5C alpha- ketoglutarate (step 3). Carbon 3 of 5C α-ketoglutarate is oxidized to CO2 and NAD+ reduced to NADH, forming 4C molecule that is attached to coA to form succinyl-coA (step 4). All 3 carbons from original pyruvate now oxidized and electrons captured by NADH. In Stage 2, highly reactive succinyl-coA is converted to oxaloacetate with ATP synthesized (step 5), electrons captured as FADH2 (step 6), and more electrons captured as NADH (step 8). Regenerated oxaloacetate now ready to react with acetyl-coA again. Two cycles of pathway will oxidize all 6 C from original glucose in glycolysis and electrons carried by NADH and FADH2 molecules will enter electron transport chain. Electron Transport and Oxidative Phosphorylation During the citric acid cycle, NAD+ (and FAD) are reduced to NADH (and FADH2) as acetyl-coA is oxidized to CO2 and oxaloacetate is regenerated. NADH and FADH2 deliver their electrons to the terminal electron transport chain which ultimately reduces molecular oxygen (O2) to H2O. NADH + ½O2 + H+ → H2O + NAD+ oxidized reduced As electrons are transferred through a series of 4 large protein complexes, protons are transferred across the mitochondrial inner membrane. to adds e- mechanical energy from spinning complex +u --ch ADP-> ATP (chemical energy) which can The unequal distribution of protons across a membrane is a source of potential energy (called the Proton Motive Force). This energy is used to generate ATP. site of glycolysis (cytoplasm) site of TCA cycle + (matrix) + + + electron transport + + + (inner membrane) ++ + ++ + +++++ Proton Motive Force +++ (inter-membrane space) We will use the mitochondrion as a model to discuss electron transport but remember its correspondence to the bacterial cell Mitochondrion Gram Negative Bacterial Cell Cytoplasm Cytoplasmic membrane Outer membrane Periplasm Inter-membrane space Human body has about 100-250 g of ATP in any instant Human requires about 60-80 kg of ATP per day This amount of ATP is provided by constant recycling of ADP to synthesize new ATP (about 1021 molecules per second in humans). Most of this production is dependent on activity within the mitochondrial inner membrane. Each human carries approx 14,000 m2 of mitochondrial inner membrane Electron Flow from Glycolysis and TCA cycle 1. NADH delivers electrons to Complex I then to Ubiquinone (Coenzyme Q). -> Part ofTCA cycle - succinate tofunerate 2. The FADH2 embedded in (Coenzyme Q) Complex II also reduces Ubiquinone. e- reduces Ubiquinone Note: Complex II is really succinate dehydrogenase from TCA cycle. 3. Mobile carrier Ubiquinol (reduced Ubiquinone) delivers electrons to Complex III. 4. Protein Cytochrome c shuttles electrons to Complex IV. 5. Electrons reduce O2 to H2O. reduce most of the TCA enzymes are floating in the mitochondrial matrix - but succinate dehydrogenase is in the inner membrane Succinate dehydrogenase (Complex II) is a physical linkage between TCA cycle and Electron Transport Chain  Ubiquinone → Ubiquinol PMF Complexes I, III, and IV pump protons across membrane as electrons pass thru NADH + ½O2 + H+ → H2O + NAD+ Free energy of transfer = ~220 kJ/mol Energy is released as e- spontaneously transfer to successively stronger oxidizers. Standard Reduction Potentials of some half reactions per mol Glucose reduction I potential Oxidant Reductant n Eo’(V) Glycolysis 2 ATP NAD+ NADH 2 -0.32 2 NADH NADP+ NADPH 2 -0.32 Pyruvate Lactate 2 -0.19 TCA cycle FAD+ FADH2 2 0 2 ATP or GTP Ubiquinone (Q) Ubiquinol (QH2) 2 0.04 2 FADH2 NO3- (nitrate) NO2- (nitrite) + H2O 2 0.42 8 NADH Fe3+ Fe2+ 1 0.77 O2 H2 O 2 0.82 Eo’ is the standard reduction potential and n is the number of electrons transferred. Eo’ refers to the half reaction, oxidant + electron to reductant. Note that e- will move NADH + ½O2 + H+ → H2O + NAD+ spontaneously to acceptors Free energy of transfer = ~220 kJ/mol with higher (more positive) reduction potentials ΔGo’ = -nFΔEo’ anaerobic respiration O2 ↳ same as acrobic but not ΔGo’ = -2(96.48 kJmol-1V-1)(0.82 V - (-0.32 V)) where, n = number of electrons transferred ΔGo’ = -2(96.48 kJmol-1V-1)(1.14 V) F, Faraday constant = 96.48 kJmol-1V-1 ΔGo’ = -220 kJ/mol ΔEo’ = Eo’ (acceptor) - Eo’ (donor) energy released as electrons travel from NADH to O2 = 220 kJ/mol NADH, enough energy to drive synthesis of about 2.5 mol ATP (from ADP and Pi). NADH → FMN → Fe-S → Q → heme b → Fe-S → heme c1 → cyt c → heme a → heme a3 → O2 -0.32 -0.30 +0.04 +0.07 +0.23 +0.25 +0.29 +0.55 +0.82 The Nature of Complexes I, II, III, IV Complex Name # of subunits prosthetic e- carriers I NADH-Q reductase 45 FMN, Fe-S cluster II Succinate dehydrogenase 4 FAD, hemes, Fe-S cluster III Q-Cytochrome c reductase 11 hemes, Fe-S cluster IV Cytochrome c oxidase 13 Cu, hemes Ubiquinone Major Electron Carrier - mobile, soluble Badenin NAD+ NAD+ + H+ + 2 e- → NADH (Nicotinamide Adenine Dinucleotide) Precursor for Nicotinamide = vitamin Niacin (B3) Major Electron Carriers - non-mobile, protein prosthetic groups isoalloxazine FAD (Flavin Adenine Dinucleotide) Precursor for Flavin = vitamin Riboflavin (B2) Fe-S clusters Fe3+ → Fe2+ (ox) (red) sometimes Histidines rather than Cysteines participate in coordination of cluster Heme groups (embedded within proteins) Fe3+ → Fe2+ COO- (ox) (red) some heme groups also include a Cu atom Heme containing proteins are called cytochromes e.g. cyt b cyt c1 cyt a Protein cyt a3 +H 3N COO- H2 C Protein +H 3N - be shuttling - complex 2 mobile electron carriers Ubiquinone (coenzyme Q) - hydrophobic molecule Cytochrome c - protein + heme Cytochrome c proteins -  Complexes I, III, IV are proton pumps energy release from electron transfer down reduction potential slope is coupled to proton transfer across mitochondrial inner membrane or in the case of bacterial cell: H+ H+ transfer from the cytoplasm into the H+ H+ periplasm. Complex I - NADH-Ubiquinone Reductase Complex electrons from NADH (produced in TCA cycle are transferred through a series of FMN and [Fe-S] clusters to oxidized ubiquinone (Q). electrons (with 2 H+ abstracted from mitochondrial matrix) reduce Q to ubiquinol (QH2). 4 H+ are pumped from the mitochondrial matrix to the inter-membrane space. QH2 is hydrophobic. Passes e- thru membrane to Complex III. note that energy released thru e- transfer has been converted to: first, mechanical energy and then a chemical/ electrical gradient. Complex II - Succinate Dehydrogenase Complex electrons from FADH2 → Fe-S → heme → Q (forming QH2) via Complex II Complex II is not a proton pump. Complex II is succinate dehydrogenase from the TCA cycle Ubiquinone (Coenzyme Q) Ubiquinone (Q) picks up electrons from Complexes I and II reduced to Ubiquinol (QH2) Complex III - Q-Cytochrome c Reductase Complex electrons from ubiquinol (QH2) transferred to a series of heme prosthetic groups and Fe-S clusters in Complex III Complex III is a proton pump. Cytochrome c the second mobile carrier of electrons in the electron chain unlike ubiquinone, Cytochrome c is a protein and is present in the inter-membrane space (not the membrane itself) Cytochrome c structure Complex IV - Cytochrome c Oxidase Complex Complex IV is the site where O2 is reduced to H2O. Electrons are delivered to Complex IV via the protein Cytochrome c. Complex IV is a proton pump. 4 protons are consumed in reduction of O2 and 4 protons are pumped across membrane. e- Cytochrome c 4 H+ cu -> Cu inter-membrane space inner O2 membrane 4 H+ 2 H 2O enator mitochondrial metabolic pathway matrix  Electron pathway from Cytochrome c to O2. Each Cytochrome c delivers one electron. Fe3+ → Fe2+ Cu2+ → Cu+ The Oxidation of Glucose and other food molecules generates a proton gradient called the Proton Motive Force. It is a electrochemical gradient that constitutes potential energy. The dissipation of this electro-chemical gradient is used to generate ATP. Protons flowing down their concentration gradient powers the motor protein ATP synthase that catalyzes the reaction: ADP + Pi → ATP PMF- protonmative Force net +2 ATP, substrate level phosphorylation Glycolysis +2 ATP, substrate level phosphorylation TCA net +26 ATP, oxidative phosphorylation ETC Inhibitors of Electron Transport and Oxidative Phosphorylation Two major types of inhibitors: Uncouplers dissipate the proton motive force (next day) Electron Flow inhibitors block electron flow usually by reacting with prosthetic groups.  theyre doing in what Electron Flow Blockers mirimichi river get to rid of invasive Complex I