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Bioenergetics

Bioenergetics. Study of energy transformations in living organisms Thermodynamics 1st Law: Conservation of E Neither created nor destroyed Can be transduced into different forms 2nd Law: Events proceed from higher to lower E states Entropy (disorder) always increases

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Bioenergetics

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  1. Bioenergetics • Study of energy transformations in living organisms • Thermodynamics • 1st Law: Conservation of E • Neither created nor destroyed • Can be transduced into different forms • 2nd Law: Events proceed from higher to lower E states • Entropy (disorder) always increases • Universe = system + surrounds

  2. Bioenergetics (E content of system) H = (useful free E) G + (E lost to disorder) TS • Gibbs Free Energy: G = H - TS • If G = negative, then rxn is exergonic, spontaneous • If G = positive, then rxn is endergonic, not spontaneous • Standard conditions (ΔG°’): 25oC, 1M each component, pH 7, H2O at 55.6M

  3. Bioenergetics A + B <--> C + D • Rate of reaction is directly proportional to concentration of reactants • At equilibrium, forward reaction = backward reaction k1[A][B] = k2[C][D] • Rearrange: k1/k2 = ([C][D])/([A][B]) = Keq • Relationship between ΔG°’ and K’eq is: G°’ = -2.303 * R * T * log K’eq If Keq >1, G°’ is negative, rxn will go forward If Keq <1, G°’ is positive, rxn will go backward

  4. ΔG°’ is a fixed value at standard conditions • ΔG under actual cellular conditions can be different • e.g., for ATP hydrolysis inside a cell, can approach ΔG = -12 kcal/mol • We will work with ΔG°’ values

  5. Coupling endergonic and exergonic rxns Glutamic acid (Glu) + NH3 --> Glutamine (Gln) G°’=+3.4 kcal/mol ATP --> ADP + Pi G°’=-7.3 kcal/mol ---------------------------------------------------------------------------------------- Glu + ATP + NH3 --> Gln + ADP + Pi G°’=-3.9 kcal/mol Glutamyl phosphate is the common intermediate

  6. ATP --> ADP + Pi ΔG°’= -7.3 kcal/mol ADP + Pi --> ATP ΔG°’= +7.3 kcal/mol C(diamond) + O2 --> CO2 ΔG°’= -94.8 kcal/mol PEP --> pyruvate + Pi ΔG°’= -14.8 kcal/mol C(graphite) + O2 --> CO2 ΔG°’= -94.1 kcal/mol P-creatine --> creatine + Pi ΔG°’= -11.0 kcal/mol G6-P --> glucose + Pi ΔG°’= -3.0 kcal/mol 1,3-BPG --> 3PG + Pi ΔG°’= -12.5 kcal/mol ---------------------------------------------------------------------------------------- What is ΔG°’ of: PEP + ADP --> pyruvate + ATP ΔG°’= -7.5 ---------------------------------------------------------------------------------------- What is ΔG°’ of: G6-P + ADP --> glucose + ATP What is ΔG°’ of: P-creatine + ADP --> creatine + ATP What is ΔG°’ of: C(s, diamond) --> C(s, graphite) ?

  7. Equilibrium vs steady state • Cells are open systems, not closed systems • O2 enters, CO2 leaves • Allows maintenance of reactions at conditions far from equilibrium O2

  8. Req’d in small amounts • Not altered/consumed in rxn • No effect on thermodynamics of rxn • Do not supply E • Do not determine [product]/[reactant] ratio (Keq) • Do accelerate rate of reaction (kinetics) • Highly specific for substrate/reactant • Very few side reactions (i.e. very “clean”) • Subject to regulation No relationship between G and rate of a reaction (kinetics) Why might a favorable rxn *not* occur rapidly? Biological Catalysts

  9. Overcoming the activation energy barrier (EA) • Bunsen burner: CH4 + 2O2 --> CO2 + 2H2O • The spark adds enough E to exceed EA (not a catalyst) • Metabolism ‘burning’ glucose • Enzyme lowers EA so that ambient fluctuations in E are sufficient

  10. Overcoming the activation energy barrier (EA) • Bunsen burner: CH4 + 2O2 --> CO2 + 2H2O • The spark adds enough E to exceed EA • Metabolism ‘burning’ glucose • Enzyme lowers EA so that ambient fluctuations in E are sufficient Catalyst shifts EA line to left <---

  11. How to lower EA • The curve peak is the transition state (TS) • Enzymes bind more tightly to TS than to either reactants or products

  12. How to lower EA • Mechanism: form an Enzyme-Substrate (ES) complex at active site

  13. How to lower EA • Mechanism: form an Enzyme-Substrate (ES) complex at active site • Orient substrates properly for reaction to occur • Increase local concentration • Decrease potential for unwanted side reactions

  14. How to lower EA • Mechanism: form an Enzyme-Substrate (ES) complex at active site • Enhance substrate reactivity • Enhance polarity of bonds via interaction with amino acid functional groups • Possibly form covalent bonded intermediates with amino acid side chains

  15. How to lower EA • Possibly form covalent bonded intermediates with amino acid side chains • Serine protease mechanism:

  16. How to lower EA • Possibly form covalent bonded intermediates with amino acid side chains • Serine protease mechanism:

  17. How to lower EA • Mechanism: form an Enzyme-Substrate (ES) complex at active site • Induce bond strain • Alter bonding angles within substrate upon binding • Alter positions of atoms in enzyme too: Induced fit

  18. Induced fit

  19. Induced fit

  20. Enzyme kinetics: The Michaelis-Menten Equation S <--> P At low [S], rate/velocity is slow, idle time on the enzyme At very high [S], rate/velocity is maximum (Vmax), enzyme is saturated V = Vmax [S]/([S] + Km) Km = [S] at Vmax/2 A low Km indicates high enzyme affinity for S (0.1mM is typical)

  21. Enzyme kinetics: pH and temperature dependence

  22. Enzyme inhibitors • Irreversible • Form a covalent bond to an amino acid side chain of the enzyme active site • Block further participation in catalysis • Example: penicillin • Inhibits Transpeptidase enzyme required for bacterial cell wall synthesis • Weak cell wall = cell burst open penicillin

  23. Enzyme inhibitors • Example: ritonavir • Inhibits HIV protease ability to process virus proteins to mature forms • Reversible • Competitive • bind at active site • Steric block to substrate binding • Km increased • Vmax not affected (increase [S] can overcome)

  24. Enzyme inhibitors • Example: anandamide (endogenous cannabinoid) • Inhibits 5-HT3 serotonin receptors that normally increase anxiety • Reversible • Noncompetitive • Do not bind at active site • Bind a distinct site and alter enzyme structure reducing catalysis • Km not affected • Vmax decreased, (increase [S] cannot overcome) Noncompetitive Competitive

  25. Drug discovery • Average cost to market ~ $1B • Average time to market ~13 years • Size of market ~ $289B per year in US (2006) • S. aureus infections are a problem in hospital settings • Drug targets • Metabolic rxns specific to bacteria • Sulfa drugs (folic acid biosynthesis) • Cell wall synthesis • Penicillin, methicillin, vancomycin • DNA replication, transcription, translation • Ciprofloxacin (DNA gyrase) • Tetracyclins (ribosome) • Zyvox (ribosome) • Introduced in 2000, resistance observed within 1 year of use

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