Mentioned the Typical Type of Bond Cleavage and Kinds of Nucleophiles and Electrophiles

Chemical Reactivity First, we mentioned the typical type of bond cleavage and kinds of nucleophiles and electrophiles commonly used by the cell in biochemical reactions 1 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) 2 Chemical Reactivity C–C bonds Examples of Nucleophilic Carbon-Carbon Bond Formation and Cleavage Reactions Ketose sugar Acetyl-CoA – OH – OH – – RR22 R2 R2 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) 3 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 Esters & Thioesters •Hydrolysis of thioesters is strongly favorable. –EXAMPLE: Acetyl-CoA is an important donor of acyl groups. –feeding two-carbon units into metabolic pathways –synthesis of fatty acids b-Mercaptoethyamine Phosphate Pantothenic Acid Pantothenic Acid Adenine Ribose Coenzyme A 4 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) Chemical Reactivity Addition-Elimination Elimination without Cleavage Enzymes will do this stereo-specifically 5 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) Chemical Reactivity Rearrangements Isomerizations and Eliminations: (No Change in Oxidation State) Proline racemase Ribulose-5phosphate epimerase 6 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) Chemical Reactivity Group Transfers • Proton transfer, very common • Methyl transfer, various biosyntheses • Acyl transfer, biosynthesis of fatty acids • Glycosyl transfer, attachment of sugars • Phosphoryl transfer, to activate metabolites ‒ also important in signal transduction ‒ proceeds through a nucleophilic substitution mechanism O W O O Z Example of ATP hydrolysis Sometimes called “bi-pyramidal” Substitution from sp3 phosphorous proceeds via the nucleophilic substitution (usually associative, SN2-like) mechanism. The nucleophile forms a partial bond to the phosphorous center, giving a pentacovalent intermediate or a penta-coordinated transition state. 7 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 ‒Nucleophilic substitution mechanism ‒Addition–elimination mechanism o Hydrolysis and Condensation reactions o Eliminations (without cleavage) •Internal rearrangements •Group transfers (H+, CH3+, PO32–) –Nucleophilic substitution mechanism •Oxidations-reductions (e– transfers) Chemical Reactivity Re-dox Lessons from Quantum Chemistry • Most organic molecules, including the reduced fuels, are in the singlet spin state. – All electrons are paired. • Molecular oxygen is in the triplet spin state. – Two electrons are unpaired. • Direct electron transfer from a singlet reduced species to a triplet-oxidizing species is quantum-mechanically unfavorable. • This is why spontaneous oxidation of biomolecules (carbon oxidation states differ by 2e–) does not occur readily, despite the exergonic reaction thermodynamics. • Cofactors, such as transition-metal ions and flavin adenine dinucleotide (FAD), are able to catalyze consecutive singleelectron transfers needed for utilization of O2. 8 Chemical Reactivity Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation. Most energy Re-dox Relative DG°’ of oxidation per carbon (kcal/mol) –196 –168 –125 –68 Least energy Chemical Reactivity Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation. Most energy 0 Re-dox Relative DG°’ of oxidation per carbon (kcal/mol) –196 –168 –125 –68 Least energy 0 9 Chemical Reactivity Re-dox EXAMPLE: Reversible Oxidation of a Secondary Alcohol to a Ketone • Many biochemical oxidation-reduction reactions involve transfer of two electrons. • In order to keep charges in balance, proton transfer often accompanies electron transfer. • In many dehydrogenases, the reaction proceeds by a stepwise transfers of proton (H+) and hydride (:H–). NAD+ NADH + +H+ :H– + H :H– + H+ + NAD+ NADH + H Chemical Reactivity Re-dox Common Redox Cofactors: NADH, NADPH, and FADH2 • Niacin Cofactors allow Hydride (2 e–) Electron Transfers • Flavin Cofactors allow single Electron Transfers Vitamin B3 (niacin) • Measure the change of absorbance at 340 nm • Very useful signal when studying the kinetics of NAD-dependent dehydrogenases What about FAD? Vitamin B2 (riboflavin) • These are commonly called pyridine nucleotides. • They can dissociate from the enzyme after the reaction. • In a typical biological oxidation reaction, hydride from an alcohol is transferred to NAD+, giving NADH. • Permits the use of molecular oxygen as an ultimate electron acceptor – flavin-dependent oxidases • Flavin cofactors are covalently or tightly bound to proteins. 10 Bioenergetics Energetics of Biochemical Reactions: Bioenergetics Energetics of Some Chemical Reactions: • Hydrolysis reactions tend to be strongly favorable (spontaneous). – Recall that being thermodynamically favorable is not the same as being kinetically rapid. • Isomerization reactions have smaller free-energy changes. – isomerization between enantiomers: DG° = 0 • Complete oxidation of reduced compounds (redox reactions) is strongly favorable. – This is how heterotrophs (sometimes called chemotrophs) obtain most of their energy. – In biochemistry, the oxidation of reduced fuels with O2 is stepwise and controlled. 11 Bioenergetics Big Picture: Flow of Electrons Generally exergonic ADP ATP Separate pools of redox cofactors Generally endergonic Bioenergetics Reduction Potential determines the Flow of Electrons Aox + Bred ⇌ Ared + Box • Reduction potential (E) – affinity for electrons; higher E, higher affinity – electrons transferred from lower E to higher E DE°’ = E°’ (e− acceptor) – E°’ (e− donor) (that which is reduced) (that which is oxidized) DE°’ = (RT / nF )ln (Keq) = –DG°’ /nF DG°’ = –n F DE°’ – For negative DG, need positive DE E°’(acceptor) > E°’(donor) 12 Bioenergetics Standard Reduction Potential (Eo’) determines the Flow of Electrons TABLE 13-7a Standard Reduction Potentials of Some Biologically Important HalfReactions Half-reaction 1/ 2 E'˚ (V) O2 + 2H+ + 2e– à H2O 0.816 Fe3+ + e– à Fe2+ NO3– + 2H+ + 2e– 0.771 à NO2– + H2O 0.421 Cytochrome f (Fe3+) + e– à cytochrome f (Fe2+) Fe(CN)63– (ferricyanide) + e– à Fe(CN)64– Cytochrome a3 (Fe3+) + e– à cytochrome a3 0.365 0.36 (Fe2+) O2 + 2H+ + 2e– à H2O2 0.35 0.295 Cytochrome a (Fe3+) + e– à cytochrome a (Fe2+) 0.29 Cytochrome c (Fe3+) + e– à cytochrome c (Fe2+) 0.254 Cytochrome c1 (Fe3+) + e– à cytochrome c1 (Fe2+) Cytochrome b (Fe3+) + e– à cytochrome b (Fe2+) Ubiquinone + 2H+ + 2e– à ubiquinol Fumarate2– + 2H+ + 2e– à succinate2– 2H+ + 2e– à H2 (at standard conditions, pH 0) Crotonyl-CoA + 2H+ + 2e– à butyryl-CoA Oxaloacetate2– + 2H+ + 2e– à malate2– 0.22 0.077 0.045 0.031 0.000 –0.015 –0.166 Source: Data mostly Loach, –in Handbook of Biochemistry and Molecular Biology, 3rd edn (G. D. Fasman, ed.), Physical –+ Pyruvate 2H +from 2e–R.àA.lactate –0.185 and Chemical Data, Vol. 1, p. 122, CRC Press, 1976. aThis is the value for free+ FAD; FAD bound to a specific flavoprotein (e.g., succinate dehydrogenase) has a different E'˚ that–0.197 Acetaldehyde + 2H + 2e– à ethanol depends on its protein environment. – FAD + 2H+ + 2e à FADH2 –0.219a Glutathione + 2H+ + 2e– à 2 reduced glutathione S + 2H+ + 2e– à H2S Lipoic acid + 2H+ + 2e– à dihydrolipoic acid AT PH 7 –0.23 –0.243 –0.29 NAD+ + H+ + 2e– à NADH –0.320 NADP+ + H+ + 2e– à NADPH –0.324 Acetoacetate + 2H+ + 2e– à β-hydroxybutyrate –0.346 α-Ketoglutarate + CO2 + 2H+ + 2e– à isocitrate –0.38 2H+ + 2e– à H2 (at pH 7) –0.414 Ferredoxin (Fe3+) + e– à ferredoxin (Fe2+) –0.432 Bioenergetics Reduction Potentials with higher affinity at bottom Hates e– Loves e– 13 Bioenergetics EXAMPLE: NAD+ NADH + +H+ :H– + H :H– + H+ + NAD+ NADH + H DE°’ = E°’ (reduction) – E°’ (oxidation) = –0.32 V – (–0.19 V) = –0.13 V DG°’ = –n F DE°’ = –(2)(23.06 kcal mol-1 V-1)(–0.13 V) = +6 kcalmol-1 This is why we always ran the reaction the other way! Bioenergetics EXAMPLE: DG°’ = –678 kcal mol-1 What is the DE°’ for the complete oxidation of glucose? DG°’ = –n F DE°’ –678 kcal mol-1 = –(24)(23.06 kcal V-1mol-1)(x V) DE°’ = +1.25 V What is the reduction potential (E°’ ) for Glc à CO2? DE°’ = E°’ (reduction) – E°’ (oxidation) 1.+1.25 V = +0.82 V – (E°’ (Glc oxidation)) E°’ (Glc oxidation) = – 0.43 V 14 Summary We learned that: • The rules of thermodynamics and organic chemistry still apply to living systems • Reactions are favorable when the free energy of products is much lower than the free energy of reactants • Biochemical phosphoryl transfer reactions are favorable when: – the phosphate donors are destabilized by electrostatic repulsion – the reaction products are often stabilized by resonance • Unfavorable reactions can be made possible by chemically coupling a highly favorable reaction to the unfavorable reaction • Oxidation-reduction reactions commonly involve transfer of electrons from reduced organic compounds to specialized redox cofactors – Reduced cofactors can be used in biosynthesis, or may serve as a source of energy for ATP synthesis. 15