Bioenergetics Study of energy transformations in living organisms Thermodynamics –1st Law: Conservation of E Neither created nor destroyed Can be transduced.

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

Bioenergetics(E content of system) H = (useful free E) G + (E lost to disorder) TS

• Gibbs Free Energy: G = H - TS– 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

BioenergeticsA + 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 forwardIf Keq <1, G°’ is positive, rxn will go backward

• 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

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

Coupling endergonic and exergonic rxns

ATP --> ADP + Pi ΔG°’= -7.3 kcal/molADP + Pi --> ATP ΔG°’= +7.3 kcal/molC(diamond) + O2 --> CO2 ΔG°’= -94.8 kcal/molPEP --> pyruvate + Pi ΔG°’= -14.8 kcal/molC(graphite) + O2 --> CO2 ΔG°’= -94.1 kcal/molP-creatine --> creatine + Pi ΔG°’= -11.0 kcal/molG6-P --> glucose + Pi ΔG°’= -3.0 kcal/mol1,3-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 + ATPWhat is ΔG°’ of: P-creatine + ADP --> creatine + ATPWhat is ΔG°’ of: C(s, diamond) --> C(s, graphite)

?

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

1) Req’d in small amounts2) Not altered/consumed in rxn3) No effect on thermodynamics of rxn

a) Do not supply Eb) Do not determine [product]/[reactant]

ratio (Keq)c) Do accelerate rate of reaction (kinetics)

4) Highly specific for substrate/reactant5) Very few side reactions (i.e. very “clean”)6) Subject to regulation

No relationship between G and rate of a reaction (kinetics)

Why might a favorable rxn *not* occur rapidly?

Biological Catalysts

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

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 <---

How to lower EA

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

How to lower EA

• Mechanism: form an Enzyme-Substrate (ES) complex at active site

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

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

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

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

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

Induced fit

Induced fit

S <--> PAt low [S], rate/velocity is slow, idle time on the enzymeAt 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)

Enzyme kinetics: The Michaelis-Menten Equation

Enzyme kinetics: pH and temperature dependence

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

Enzyme inhibitors• Reversible

– Competitive• bind at active site• Steric block to substrate binding

– Km increased– Vmax not affected (increase [S] can overcome)

• Example: ritonavir– Inhibits HIV protease ability to process virus proteins to mature forms

Enzyme inhibitors• 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)

NoncompetitiveCompetitive

• Example: anandamide (endogenous cannabinoid)– Inhibits 5-HT3 serotonin receptors that normally

increase anxiety

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