Energy Coupling, Chemical Reactivity, Bioenergetics

Biochemistry II OUTLINE Review of 421 Goals of 422 Review of chemical principles Thermodynamics C/O cycles Overview of Metabolism ATP cycles Energy Coupling Chemical Reactivity Bioenergetics Metabolism THE ATP CYCLE This “buffering” of energy in the cell keeps the [ATP] high Energy enough to keep fighting the second law of thermodynamics. Charge = + ½ [ADP-3] [ATP-4] + [ADP-3] + [AMP-2] [ATP-4] 1 Metabolism –22– – + + HPO HPO + H 4 44 + H 2PO (at pH 7) + H+ + H2O Adenosine diphosphate (ADP) ADP-3 ATP-4 (at pH 7) If this reaction is allowed to come to equilibrium, what is the DG? the actual free-energy change of a reaction depends on: – A constant (the standard change in free energy, DG°) – actual concentrations of products and reactants – For the reaction aA + bB ⇌ cC + dD: c d [C ] [ D] DG′= DG'°′ ° + RT ln [ A]a [ B]b [In biochemistry, we add a prime (′) because we pull [H+] out of the expression and set it to 10-7, not 1 M] Metabolism + HPO4–2 + H+ + H2O (at pH 7) Adenosine diphosphate (ADP) c d [C ] [ D] DG′= DG'°′ ° + RT ln [ A]a [ B]b [C ]c [ D]d DG′= DG°′ '° + RT ln K′eqa b d ] [ B] [C ]c [ D[]A = DG = DG°′ '° + RT ln K′eqa b [ A] [ B] TABLE 13-2 Relationship between Equilibrium Constants and Standard FreeEnergy Changes of Chemical Reactions DG' DG°′= DG°ʹ= –7.3 kcal/mole kcal/mole (kJ/mol) 103 –17.1 102 –11.4 –2.7 101 –5.7 –1.4 0.0 0.0 1 TABLE 13-3 Relationships among K'eq, DG'˚, and the Direction of Chemical Reactions When K'eq is . . . >1.0 1.0 <1.0 DG'˚ is . . . Starting with all components at 1 M, the reaction . . . negative proceeds forward zero is at equilibrium positive proceeds in reverse 10–1 But, what is the actual DG′ of ATP Hydrolysis IN THE CELL? (kcal/mol)a K'eq –4.1 5.7 1.4 10–2 11.4 2.7 10–3 17.1 4.1 10–4 22.8 5.5 10–5 28.5 6.8 10–6 34.2 8.2 a Although joules and kilojoules are the standard units of energy and are used throughout this text, biochemists and nutritionists sometimes express DG'˚ values in kilocalories per mole. We have therefore included values in both kilojoules and kilocalories in this table and in Tables 13-4 and 13-6. To convert kilojoules to kilocalories, divide the number of kilojoules by 4.184. 2 Metabolism + HPO4–2 + H+ + H2O (at pH 7) Adenosine diphosphate (ADP) c Go back the original equation: DG = DG °'+ RT ln DG′ = DG°′ [MgADP- ] × [Pi ] [MgATP2- ] At 25 °C: DG°′ of ATP Hydrolysis is Mg++ Dependent d [C ] [ D] DG′= DG'°′ ° + RT ln [ A]a [ B]b DG′ = DG°′ [ADP-3] [HPO4-2] + RT ln [ATP-4] [ADP-3] [HPO4-2] + 0.59ln [ATP-4] In RBCs = 1.5 x 10-4 M DG′ = DG°′ + 1.36log (1.5 x 10-4) DG′ = (-7.3) + (-5.2) DG′ = -12.5 kcal/mole WOW! Other than concentrations, what makes the DG′ of ATP so high? Metabolism • Contributions of the incredible favorability of the ATP hydrolysis reaction: – Better charge separation in products (relief of charge repulsion) – More favorable resonance stabilization of products ❸ H2PO4– – Ionization of products (ADP or Pi) ⇌ HPO + H 42– + (at pH 7) Are there other compounds in the cell with high negative DG°′ of hydrolysis? 3 Metabolism • Contributions of the incredible favorability of the ATP hydrolysis reaction: – Better charge separation in products (relief of charge repulsion) – More favorable resonance stabilization of products ❸ H2PO4– – Ionization of products (ADP or Pi) ⇌ HPO + H 42– + (at pH 7) Are there other compounds in the cell with high negative DG°′ of hydrolysis? Metabolism DG°′= –12 kcal/mole TABLE 13-4 Standard Free-Energy Changes of Some Chemical Reactions D G'˚ Reaction type 1,3-bisphosphoglycerate DG°′= –11.8 kcal/mole DG°′= –10.3 kcal/mole (kJ/mol) (kcal/mol) Hydrolysis reactions Acid anhydrides Acetic anhydride + H2O à 2 acetate ATP + H2O à ADP + Pi ATP + H2O à AMP + PPi PPi + H2O à 2Pi UDP-glucose + H2O à UMP + glucose 1-phosphate –91.1 –30.5 –45.6 –19.2 –43.0 –21.8 –7.3 –10.9 –4.6 –10.3 Esters Ethyl acetate + H2O à ethanol + acetate Glucose 6-phosphate + H2O à glucose + Pi –19.6 –13.8 –4.7 –3.3 Amides and peptides Glutamine + H2O à glutamate 1 NH4+ Glycylglycine + H2O à 2 glycine –14.2 –9.2 –3.4 –2.2 Glycosides Maltose + H2O Lactose + H2O –15.5 –15.9 –3.7 –3.8 –7.3 –1.7 –1.7 –0.4 à à 2 glucose glucose + galactose Rearrangements Glucose 1-phosphate à glucose 6-phosphate Fructose 6-phosphate à glucose 6-phosphate Elimination of water Malate à fumarate + H2O Oxidations with molecular oxygen Glucose + 6O2 à 6CO2 + 6H2O Palmitate + 23O2 à 16CO2 + 16H2O 3.1 0.8 –2,840 –9,770 –686 –2,338 DG°′= –14.8 kcal/mole = –61.9 kJ/mole (PEP) 4 Metabolism Phosphates: Ranking by the Standard Free Energy of Hydrolysis Phosphate can be transferred from compounds with higher ΔG°′ to those with lower ΔG°′. Metabolism TABLE 13-5 Total Concentrations of Adenine Nucleotides, Inorganic Phosphate, and Phosphocreatine in Some Cells Concentration (mM)a ATP ADPb AMP Energy Charge Pi Rat hepatocyte 3.38 1.32 0.29 4.8 0 Rat myocyte 8.05 0.93 0.04 0.81 0.94 0.87 0.94 0.86 8.05 28 2.72 4.7 Rat neuron 2.59 0.73 0.06 Human erythrocyte 2.25 0.25 0.02 E. coli cell 7.90 1.04 0.82 PCr 1.65 0 7.9 0 a For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and mitochondria). In the other types of cells the data are for the entire cell contents, although the cytosol and the mitochondria have very different concentrations of ADP. PCr is phosphocreatine, discussed on p. 516. b This value reflects total concentration; the true value for free ADP may be much lower (p. 509). Cellular ATP concentration is usually far above the equilibrium concentration, making ATP a very potent source of chemical energy. 5 Energy Coupling Energy Coupling • Question is how do cells use these “high-energy compounds” to ensure that thermodynamically unfavorable reactions can become favorable? • Chemical coupling of exergonic and endergonic reactions allows otherwise unfavorable reactions. • TWO basic mechanisms: – e.g., The hydrolysis of the “high-energy” molecule (ATP), which is exergonic, is coupled with an otherwise endergonic reaction during an enzyme mechanism. – e.g., a series of otherwise endergonic reactions during a metabolic pathway are linked in the reaction series to an overwhelming exergonic reaction (all catalyzed by enzymes). 6 Energy Coupling Enzymatic reaction These two reactions take place at the same place (active site) Replace the H–O–H with H–O–R (glucose) + Glucose Kinase (aka Hexokinase) Glucose 6-phosphate Energy Coupling Two-Step Group Transfers Facilitate ATP-Dependent Reactions 7 Energy Coupling Standard free-energy changes are additive: (1) A (2) B Sum: A B ΔG1 C ΔG2 C ΔG1 + ΔG2 Coupling exergonic and endergonic reactions: SgPgQgRgZ Net: S P Q R S P Q R Z Z DG = +12 kcal/mole DG = +1 kcal/mole DG = -4 kcal/mole DG = -14 kcal/mole DG = -5 kcal/mole Chemical Reactivity First lets quickly review bond cleavage and nucleophilicity in terms of biochemical instances 8 Chemical Reactivity Chemistry at Carbon: Homolytic versus Heterolytic Cleavage • Covalent bonds can be broken in two ways. • Homolytic cleavage is rare in biochemistry, but makes up some of the most interesting reactions. • Heterolytic cleavage is common, but the products are highly unstable, dictating the chemistry that occurs: mostly carbanion chemistry Chemical Reactivity Chemistry at Carbon: Homolytic versus Heterolytic Cleavage • Covalent bonds can be broken in two ways. • Homolytic cleavage is rare in biochemistry, but makes up some of the most interesting reactions. • Heterolytic cleavage is common, but the products are highly unstable, dictating the chemistry that occurs: mostly carbanion chemistry 9 Chemical Reactivity Nucleophiles and Electrophiles in Biochemistry Definition: -phile (suffix, Latin): to love, or demonstrate fondess towards • Nucleophiles love nuclei. The nuclei of an atom contains positively charged particles (protons), so nucleophiles love to react with positive charges and are electron rich. • Electrophiles love electrons. Electrons are negatively charged, so electrophiles must be positively charged. Chemical Reactivity Nucleophiles and Electrophiles in Biochemistry Definition: -phile (suffix, Latin): to love, or demonstrate fondess towards • Nucleophiles love nuclei. The nuclei of an atom contains positively charged particles (protons), so nucleophiles love to react with positive charges and are electron rich. • Electrophiles love electrons. Electrons are negatively charged, so electrophiles must be positively charged. 10 Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) •Oxidations-reductions (e– transfers) Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) •Oxidations-reductions (e– transfers) 11 Chemical Reactivity C–C bonds Examples of Nucleophilic Carbon-Carbon Bond Formation and Cleavage Reactions Chemical Reactivity Most biochemical reactions fall within only a few categories: •Cleavage and formation of C–C bonds •Cleavage and formation of polar covalent bonds (Addition-Elimination Reactions) ‒nucleophilic substitution mechanism ‒addition–elimination mechanism o hydrolysis and condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) •Oxidations-reductions (e– transfers) 12 Chemical Reactivity Addition-Elimination • Substitution from sp3 carbon proceeds normally via the nucleophilic substitution (SN1 or SN2) mechanism. • Substitution from the sp2 carbon proceeds normally via the nucleophilic addition– elimination mechanism. – nucleophile adds to the sp2 center, giving a tetrahedral intermediate – leaving group eliminates from the tetrahedral intermediate – leaving group may pick up a proton Chemical Reactivity Addition-Elimination Hydrolysis of Thioesters •Hydrolysis of thioesters is strongly favorable. –such as acetyl-CoA •Acetyl-CoA is an important donor of acyl groups. –feeding two-carbon units into metabolic pathways –synthesis of fatty acids 13 Chemical Reactivity Addition-Elimination Elimination without Cleavage Enzymes will do this stereo-specifically 14