Welcome to Class 8! Introductory Biochemistry! Announcements / Reminders! Midterm TA led Review Sessions!

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1 Announcements / Reminders Midterm TA led Review Sessions Welcome to Class 8 Sunday, February 23 from 8-10pm Location: Science Center Main Room (315) Office Hours Prof Salomon: SFH 270 on Thursday Feb 20, 2:30-4:30 TAs: by appointment Introductory Biochemistry Sapling problem set 3 due Monday, February 24, 5pm Midterm 1 is Tuesday February 25 at 1 pm Location: Last names beginning with A-M Macmillan 117 Last names beginning N-Z BERT 130 (BERT= Building for Enviromental Research and Teaching, with greenhouse on top. Previously called Hunter labs) Energy released by hydrolysis of biological phosphate compounds figure

2 Hydrolysis of phosphocreatine ATP can provide energy by group transfer even when there is no net transfer of P Derivation of energy from ATP hydrolysis generally involves covalent participation of ATP in the reaction. Formation of glutamine by condensation of glutamate with NH 3 is endergonic (positive ΔG' º). Formation of γ-glutamyl P by transfer of P from ATP is exergonic (negative ΔG' º). Phosphocreatine has a high phosphoryl group transfer potential. It can drive the formation of ATP from ADP. figure Formation of glutamine by displacement of P from γ-glutamyl P by NH 3 is exergonic (negative ΔG' º). The net coupled reaction is exergonic (negative ΔG' º). figure Class 8: Outline and Objectives l Redox reactions l Oxidation states of carbon l Relationship between G and E l Electron carriers NADH, NADPH, FAD, FMN l Where does the energy come from? l Overview metabolism, catabolism, anabolism l Glycolysis l Pathway l Regulation l Substrate channeling l Fermentation l Lactate l Ethanol l Pentose phosphate pathway l Fructose and galactose metabolism Many enzyme reactions involve a change in the oxidation state of the substrate 2

3 e - affinity Redox Reactions stronger oxidants Electrons are transferred in reduction-oxidation reactions. Redox reactions require an electron donor (reducing agent) and an electron acceptor (oxidizing agent). Therefore, two simultaneous reactions occur in a redox process. stronger reductants The reduction potentials of redox half-reactions under standard conditions can be tabulated, like ΔG' º values for other reactions. The strongest oxidant is O 2 and the strongest reductant is a small Feprotein called ferredoxin. stronger oxidants Measurement of the standard reduction potential of a redox pair stronger reductants The E' º value is for a half reaction under standard conditions (all components at 1 M except H + and H 2 O). The units of E' º is volts (V). Standard reduction potentials, E' º, are relative to that of hydrogen (E' º 0). Figure

4 Standard Reduction Potentials E The reduction potentials of redox couples under standard conditions can be tabulated, like ΔG' º values for other reactions. The E' º value is for a half reaction under standard conditions (all components at 1 M except H + and H 2 O). The units of E' º is volts (V). The tabulated reduction potentials are for the reduction reaction: Oxidized form + electrons Reduced form E' º (Volts) Standard reduction potentials, E' º, are relative to that of hydrogen (E' º 0). For an actual redox reaction, a reduction must be accompanied by a oxidation so that electrons are not left over or deficient. For any complete redox reaction, ΔE' º = E' º for the substance being reduced (the oxidant) minus E' º for the substance being oxidized (the reductant). stronger oxidants stronger reductants The tabulated reduction potentials are for the reduction reaction: Oxidized form + electrons Reduced form Example: 2Fe 3+ + Ethanol 2Fe 2+ + Acetaldehyde + 2H + ΔE' º = ( 0.197)= V Note that the number of atoms or molecules involved, and the number of electrons transferred, don t enter into these calculations. Oxidation states of carbon in the biosphere C bonding electrons C bonding electrons Concentration dependence of E Like G, the reduction potential E of a half cell is concentration dependent: RT E = E' º + ln nf [electron acceptor A + ] [electron donor A] ΔE = (E 2 E 1 ) V E = E 1 V 2H + /H 2 + E = E 2 V 2A + /2A If [A + ] = [A], E = E' º. If [A + ] > [A], E > E' º. If [A + ] < [A], E < E' º. Figure increasing electronegativity: H < C < S < N < O the more electronegative atom owns the bonding electrons Example: The ratio NAD + /NADH is kept high in the cell. This makes E more positive than E' º (makes the couple a stronger oxidant than if [NAD + ] = [NADH]) and favors the reaction direction NAD + + H + NADH (ΔG is more negative than ΔG' º) 4

5 Standard Reduction Potential Differences are Mathematically Related to the Standard Free Energy for the Reaction 2Fe3+ + Ethanol 2Fe2+ + Acetaldehyde + 2H+ ΔE' º = ( 0.197) = V ΔG = nfδe n = the number of electrons transferred in the reaction F = The Faraday Constant: 96.5 kj/v-mol For the above reaction, ΔG' º = (2)(96.5 kj/v-mol)(0.968 V) = kj/mol. Note that the number of electrons exchanged does enter into the calculation of ΔG' º from ΔE' º. Note that a positive ΔE' º corresponds to a negative ΔG' º (exergonic). stronger oxidants stronger reductants Electrons are transferred by cofactors acting as electron carriers Very important biological redox cofactors are the pyridine nucleotides and flavin nucleotides. The reduced forms of these cofactors are relatively strong reductants. Pyridine Nucleotide Coenzymes Flavin nucleotide cofactors Niacin (vitamin B3) Nicotinamide Adenine Dinucleotide (NAD) Nicotinamide Adenine Dinucleotide Phosphate (NADP) NAD(P)+ = oxidized form NAD(P)H = reduced form Figure Riboflavin (vitamin B2) Nicotinamide ring Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) FMN, FAD = oxidized forms FMNH2, FADH2 = reduced forms Figure

6 Coenzymes NAD and NADP Four ways to transfer electrons l Directly as electrons (Fe 2+ + Cu 2+ Fe 3+ + Cu + ) l As hydrogen atoms (AH 2 + B A + BH 2 ) l As a hydride ion :H (CH 3 CH 3 + NAD + CH 2 =CH 2 + NADH + H + ) l Through direct combination with oxygen (RCH 3 + 1/2 O 2 RCH 2 OH) The reduced nicotinamide ring has a characteristic near-uv absorption near 340 nm, which can be used to follow the progress of the enzymatic reaction in kinetic experiments. Figure 13-24b For most biological molecules, the unit of oxidation and reduction is two reducing equivalents, i.e., two electrons, i.e., pairs of electrons are gained or lost in each redox reaction. In biological systems, oxidation is often synonymous with dehydrogenation (loss of hydrogen, note that there is no oxygen involved), i.e. gain of a double bond between carbon atoms or change of an alcohol to a carbonyl oxygen in an organic molecule. 2 Fe Fe H + Several ways of expressing redox reactions NAD + + H + + 2e NADH Overview: Catabolism and anabolism metabolism: meta = change; bolism throwing NAD + + 2[H] NADH + H + NADP + + 2[H] NADPH + H + NAD(P) + + 2[H] NAD(P)H + H + FMN + 2H + + 2e FMNH 2 ana = up, back, again reduction cata = down oxidation FAD + 2[H] FADH 2 Part II, Figure 3 6

7 Catabolic pathways converge, anabolic pathways diverge Three stages of cellular respiration Glycolysis splits sugars and partially oxidizes the products, generating substrates for complete oxidation to CO 2, and also generates some ATP and reduced cofactors. (In fermentations, the reduced cofactors are re-oxidized in non-energy-generating reactions.) Glycolysis: glyco = sugar; lysis = splitting The citric acid cycle completely oxidizes the products of glycolysis to CO 2 and generates reduced cofactors as well as some ATP. Part II, Figure 4 Respiratory re-oxidation of the reduced cofactors generated in the above stages is coupled to the synthesis of large amounts of ATP. Figure 16-1 Class 8: Outline and Objectives Oxidation states of carbon in the biosphere C bonding electrons C bonding electrons l Redox reactions l Oxidation states of carbon l Relationship between G and E l Electron carriers NADH, NADPH, FAD, FMN l Where does the energy come from? l Overview metabolism, catabolism, anabolism l Glycolysis l Pathway l Regulation l Substrate channeling l Fermentation l Lactate l Ethanol l Pentose phosphate pathway l Fructose and galactose metabolism Sugars have a composition like formaldehyde, (CH 2 O) n CH 2 O + H 2 O CO 2 + 4H + + 4e Figure

8 Fire: NOT stepwise oxidation of glucose Glycolysis: stepwise oxidation of glucose Oxygen has a higher affinity for electrons than glucose and the various electron carriers (e.g. NADH). Therefore, the transfer of electrons from these molecules to O2 is energetically favorable. Glucose + 6 O2 6 CO2 + 6 H2O G o= 2840 kj/mol = kj/mol C Only kj/mol are required to form one molecule of ATP from ADP + Pi Stepwise oxidation of glucose converts electron flow into usable energy. The energy released in some steps can be captured by coupling the step to ATP synthesis, or by temporarily storing the electrons in a molecule (e.g., NADH) whose re-oxidation can be coupled to ATP synthesis. Glycolysis: Part II Glycolysis: Part I Figure Figure

9 1 1. Formation of glucose 6-phosphate 1. Formation of glucose 6-phosphate Importance of phosphorylated intermediates: Negative charge traps intermediates inside the cell, even if the concentration outside is much lower. Phosphoryl groups conserve energy. The binding energy resulting from enzyme interactions with phosphate groups helps to lower the activation energy and increases the specificity of reactions. Glucose + Pi Glucose 6-P ΔG' 0 = 13.8 kj/mol ATP ADP + Pi ΔG' 0 = 30.5 kj/mol Glucose + ATP Glucose 6-P + ADP ΔG' 0 = 16.7 kj/mol The large negative ΔG for ATP hydrolysis drives the reaction. Coupled reactions drive endergonic processes. 2. Isomerization and second phosphorylation Enzyme-limited vs. substrate-limited reactions 2 3 Aldose Ketose Enzyme-limited reaction (far from equilibrium) Substrate-limited reactions (at or near equilibrium) For some steps in glycolysis, the substrate/product ratios are near the equilibrium ratios, because the involved enzymes are relatively fast or abundant. flux is substrate-limited. Example: steps 2, 4 For a step that is catalyzed by a relatively slow or scarce enzyme, the substrate/product ratio is greater than the equilibrium ratio. flux is enzyme-limited. Example: steps 1, 3 Lehninger 3rd Ed., Figure

10 Regulation of metabolic pathways Metabolic flux must be controlled: Regulation of metabolic pathways Most enzymes in a pathway operate near their equilibrium, but some enzymes that are stategically located operate far from equilibrium. the demand for ATP production in muscle may increase 100-fold in a few seconds in response to exercise relative proportions of carbohydrate, fat and protein in the diet vary from meal to meal the supply of fuels obtained in the diet is intermittent (between meals, starvation) Characteristics of enzymes that are regulated: They control the rate of the respective pathway and whether it is turned on or shut off. They catalyze reactions that are out of equilibrium and which are enzyme-limited (valve function). They catalyze highly exergonic (effectively irreversible) reactions, thus driving the pathway forward. They differ in a catabolic vs. anabolic pathway. The two ATP-requiring steps of glycolysis are regulated Allosteric control of enzyme activity: Hexokinase Allosteric site Glucose-bound form: induced fit Hexokinase is allosterically inhibited by its own product, glucose 6-phosphate. Figure Figure

11 PFK-1 regulation is complex 4. Aldol cleavage (the literal glycolytic reaction) ATP is a required substrate, but at high concentration, ATP can also bind to an allosteric site and inhibit PFK-1. This inhibition is relieved by AMP, which competes with ATP for binding at the allosteric site. Even though G' º > 0, the reaction proceeds in the forward direction because the reaction products are removed quickly by later steps, pulling the reaction in the direction of cleavage. G < 0. Figure 15-16b,c 5. Isomerization Triosephosphate isomerase: a perfect enzyme Ketose Aldose Limitation 1: a catalyst cannot affect the position of the reaction equilibrium TIM accelerates the isomerization by a factor of compared to the chemical reaction Even though G' º > 0, the reaction proceeds in the forward direction because the reaction products are removed quickly by later steps, pulling the reaction in the direction of cleavage. G < 0. Limitation 2: a catalyst cannot catalyze an interconversion faster than the substrate can find the enzyme in solution the k cat /K M ratio for TIM is 2 x 10 8 M -1 s -1, which is close to the diffusion limit of a small molecule in solution 11

12 Glycolysis: Summary of Part I The fate of the hexose carbon atoms Figure 14-7 In this phase, a glucose molecule is converted to two triose phosphate molecules, at the expense of (driven by) the hydrolysis of two ATP molecules. Figure Glycolysis: Part II The first substrate-level phosphorylation uses two enzymes 6 7 Figure

13 6 Oxidative Phosphorylation of Glyceraldehyde 3-P 7 The first substrate-level ADP phosphorylation 1,3-bis-P-Glycerate 3-P-Glycerate + Pi ADP + Pi ATP 1,3-bis-P-Glycerate + ADP 3-P-glycerate + ATP ΔG' 0 = 49.0 kj/mol ΔG' 0 = 30.5 kj/mol ΔG' 0 = 18.5 kj/mol The large negative ΔG' 0 for 1,3-bis-P-glycerate hydrolysis drives the reaction 3-P-glycerate + 2 H+ + 2 e Glyceraldehyde 3-P E'o = V NAD+ + H+ + 2 e NADH E'o = 0.32 V ΔG' 0 = -2(96.5)( ) ΔG' 0 = kJ/mol 3-P-glycerate + Pi 1,3-bis-P-glycerate ΔG' 0 = 50.7 kj/mol Glyceraldehyde 3-P + Pi + NAD+ 1,3-bis-P-glycerate + NADH + H+ ΔG' 0 = 6.3 kj/mol ΔG' o = nfδe' o The very large negative ΔG' 0 for glyceraldehyde 3-P oxidation drives the reaction Why is hydrolysis of 1,3-BPG so exergonic? 1,3-BPG is a high-energy compound Figure The reaction product 3-P-glyceric acid is a strong acid, which immediately ionizes at physiological ph. 2. Resonance of the ionized reaction product 3-P-glycerate distributes the negative charge over 2 oxygen atoms, which stabilizes the ionized form and effectively lowers the concentration of the immediate hydrolysis product. Figure

14 The coupled reaction [6 + 7] is overall exergonic Glyceraldehyde 3-P + ADP + P i + NAD + 3-P-Glycerate + ATP + NADH + H + Overall G' º = ( 18.5) = 12.2 kj/mol Under cellular conditions, steps 6 and 7 are reversible. 6 7 Substrate channeling between reactions 6 and 7 Protects 1,3-bisphosphoglycerate from spontaneous hydrolysis Ensures that reactions 6 and 7 remain closely coupled Lehninger 3rd Ed., Figure ,3-BPG is an intermediate in step 8 The second substrate-level phosphorylation

15 Like 1,3-BPG, PEP is a high-energy compound PEP hydrolysis PEP hydrolysis is driven by tautomerization of the immediate hydrolysis product (enol form) to the more stable keto form, which lowers the effective concentration of the immediate hydrolysis product. About half of the released energy ( G' º = 61.9 kj/mol) is captured in the formation of ATP ( G' º = 30.5 kj/mol), the rest (net G' º = 31.4 kj/mol) constitutes a driving force to pull the reaction forward. Figure Glycolysis: Summary of Part II Figure Steady-state concentrations of glycolytic intermediates in erythrocytes In this phase, the initial two ATP molecules that were hydrolyzed in Part I are regenerated, and two more ATP plus two NADH molecules are formed for each molecule of glucose. Figure Metabolite Glucose Glucose 6-P Fructose 6-P Fructose 1,6-bis-P Dihydroxyacetone-P Glyceraldehyde 3-P 1,3-bis-P-glycerate 2,3-bis-P-glycerate 3-P-glycerate 2-P-glycerate P-enol-pyruvate Pyruvate Lactate ATP ADP Pi Concentration (mm) From Minakami, S. and Yoshikawa, H Biochem. Biophys. Res. Comm. 18:

16 Calculation of the overall energetics of glycolysis The standard free energies of some of the steps of glycolysis are positive, even though the overall standard free energy is negative. At the actual cellular concentrations of the intermediates, the free energies of all of the steps are either negative or close to zero. Garrett and Grisham, Figure First, calculate the energy released on complete oxidation of pyruvate: Glucose (C 6 H 12 O 6 ) + 6 O 2 6 CO H 2 O ΔG' 0 = 2840 kj/mol 2 Pyruvate (C 3 H 4 O 3 ) + 2 NADH + 2 H + Glucose + 2 NAD + ΔG' 0 = 146 kj/mol 2 NAD H e 2 NADH E' o = V ΔG' 0 = kj/mol 2 H 2 O O H e E' o = V ΔG' 0 = kj/mol 2 Pyruvate + 5 O 2 6 CO H 2 O ΔG' 0 = kj/mol Then use the value for the complete oxidation of 2 pyruvates to calculate the overall energetics of glycolysis: Glucose + 6 O 2 6 CO H 2 O ΔG' 0 = 2840 kj/mol 6 CO H 2 O 2 Pyruvate + 5 O 2 ΔG' 0 = kj/mol 2 ADP + 2 P i 2 ATP + 2 H 2 O ΔG' 0 = 61 kj/mol 2 NAD H e 2 NADH E' o = V ΔG' 0 = kj/mol 2 H 2 O O H e E' o = V ΔG' 0 = 315 kj/mol Glucose + 2 ADP + 2 P i + 2 NAD + 2 Pyruvate + 2 ATP + 2 H 2 O + 2 NADH + 2 H + ΔG' 0 = 82 kj/mol (Values from Lehninger) Of the energy released in the partial oxidation of glucose to pyruvate, 61/2840 = 2.1% is captured as ATP. If O 2 is available and can be used, another 438.5/2840 = 15.4% is potentially obtainable from re-oxidation of NADH, and an additional /2840 = 79.4% is potentially obtainable from the complete oxidation of pyruvate. Reoxidation of NADH is critical Class 8: Outline and Objectives Glucose + 2 ADP + 2 P i + 2 NAD + 2 Pyruvate + 2 ATP + 2 H 2 O + 2 NADH + 2 H + ΔG'º = 82 kj/mol Compared to the amount of glucose that is converted to pyruvate, there are tiny amounts of NAD + in cells. Glycolysis would quickly grind to a halt unless NADH is re-oxidized to NAD +. l Redox reactions l Oxidation states of carbon l Relationship between G and E l Electron carriers NADH, NADPH, FAD, FMN l Where does the energy come from? l Overview metabolism, catabolism, anabolism l Glycolysis l Pathway l Regulation l Substrate channeling l Fermentation l Lactate l Ethanol l Pentose phosphate pathway l Fructose and galactose metabolism 16

17 Fates of Pyruvate: Aerobic and anaerobic pathways Lactic acid and alcohol fermentation Both processes regenerate NAD + to allow continued glycolysis. muscle, microorganisms muscle, microorganisms yeast yeast Fermentation and the Pasteur Effect In the absence of oxygen, the ATP generated during glycolysis is the sole energy derived from partial glucose oxidation to pyruvate. Major pathways of glucose utilization Problem that needs to be solved: NAD + has to be regenerated to allow continued glycolysis. Solution: Reduce pyruvate, e.g. to lactate or ethanol, using the NADH that was generated during glycolysis. This does not require or generate energy (ATP). Fermentation does not capture very much of the energy that is potentially available from the complete oxidation of glucose. Energy available from complete glucose oxidation: 2840 kj/mol Energy required to form two mols of ATP: 2 x 30.5 = 61 kj/mol of glucose (2.1 %). In organisms that can ferment in the absence of O 2 and respire in the presence of O 2, the rate of anaerobic glucose consumption is much higher (Pasteur effect in yeast). and NADPH Figure

18 Pentose phosphate pathway Pentose phosphate pathway - Overview Figure Figure Recycling of pentose phosphates in nonoxidative reactions Recycling of pentose phosphates in nonoxidative reactions (6) (2) (4) 4 (2) 2 (3) (2) 2 3 (2) (2) (1) (2) 2 2 Figure 14-23a Figure 14-23b 18

19 Feeder pathways (convergence) Fructose catabolism liver muscle, kidney glycerol triacylglycerides Figure glycerol-3-phosphate phospholipids 1 Galactose catabolism 1. Galactokinase phosphorylates galactose to form galactose-1-p 2 4 Phosphoglucomutase Glucose 6-phosphate 3 2. Galactose-1-P is exchanged for glucose-1- P of UDP-glucose (a sugar-nucleotide). UDP (uridine diphosphate) functions as a coenzyme-like carrier of hexose groups NADH NAD+ 3. UDP-galactose is isomerized to UDPglucose by an epimerase that contains a bound NAD +. NADH and the 4-keto-UDPsugar are enzyme-bound intermediates 4. Glucose-1-P is isomerized to glucose-6-p Figure