Bioenergetics and Biochemical Reactions

Chapter 13

Life needs energy

• Living organisms are built of complex structures

• Building complex structures (low in entropy) is only possible when energy is spent in the process

• The ultimate source of this energy on Earth is the sunlight

Metabolism sum of all chemical reactions in the cell

• Series of related reactions: metabolic pathways

• Pathways that are primarily energy-producing

– Catabolism

• Pathways using energy to build complex structures

– Anabolism or Biosynthesis

Metabolism uses a limited chemical toolset

• All metabolic reactions involve the formation or breaking of a covalent bond

• Five classes of reactions that occur in biochemistry

1. Oxidation-Reduction (REDOX)

2. Carbon-Carbon bond formation/breaking

3. Internal Rearrangements

- Isomerizations and Eliminations

4. Group Transfers

5. Free Radical Reactions

Living Systems Follow the Laws of Thermodynamics

• Living organisms cannot create energy from nothing

• Living organisms cannot destroy energy

• Living organism may transform energy from one form to another

• In the process of transforming energy, living organisms must increase the entropy of the universe

• Living organisms extract useable energy from their surroundings, and release useless energy (heat)

– cells are open systems

– living systems are never at equilibrium

DG = DH - TDS

• G: Gibbs Free Energy (J/mol) – negative – exergonic

– positive – endergonic

• H: Enthalpy (J/mol) – reflects the number and kind of bonds in reactants vs. products

– negative: exothermic (releases heat)

– positive: endothermic (takes up heat from surroundings)

• S: Entropy (J/mol*K) – positive: products are more disordered

– negative: products are more ordered

• T: Temperature (K)

Gibbs Free Energy: DG

• DG for spontaneously reacting systems is always NEGATIVE

• Source of energy for living cells & their reactions

• Measure of how far from equilibrium a system or reaction is:

– amount of work that can be done

– DG at equilibrium is 0

Living Systems are NOT at Equilibrium

• Tendency of a reaction to move towards equilibrium is a driving force

• DG represents the magnitude of this driving force

• DG is a function of the standard free energy change DG°

DG = DG° + RTln [products]/[reactants]

Biochemical Standard Free Energy Change

DG'° • Biochemical Standard conditions:

– 298 K (25°C)

– reactants & products initially at 1 M concentrations

– partial pressures of 101.3 kPa (1 atm)

– [H+] = 10-7 (pH = 7.0)

– [H2O] = 55.5 M

• Standard transformed constants: DG'° & K'eq

• DG'° and K'eq are both physical constants

– describe the difference in free energy content between reactants and products under standard conditions

– measure of driving force under physiologically relevant conditions.

DG'° and K'eq

DG'° = -RT lnK'eq

DG and DG'° are Different

• DG'° - standard free energy – is a constant physical characteristic of a reaction

• DG – actual free energy – Function of reactant & product concentrations, and temperature that may

not match standard conditions

– DG is changes as the reaction progresses

• negative in spontaneous reactions progressing towards equilibrium

• less negative as reaction proceeds (closer to equilibrium)

• 0 at the point of equilibrium

DG and DG'° are Related

• Consider a chemical reaction:

aA + bB cC +dD

DG = DG'° + RT ln [C]c [D]d

[A]a [B]b

• Actual prevailing concentrations in the system

• defined as the mass-action ratio, Q

DG = DG'° + RTlnQ

DG = DG'° + RT ln [C]c [D]d [A]a [B]b

• To find the actual free energy, DG

– enter in actual concentrations of A, B, C, D

– R, T, and DG'° are standard constant values

• When a reaction has reached equilibrium: DG'° = -RTlnK'eq

• Spontaneity depends on DG, not DG'°

– DG is a maximum for energy delivery (always thermal loss)

– does not take into account activation energy

– DG is independent of pathway by which reactions occur

• Living cells utilize catalysts (enzymes) to lower activation energies and speed up reaction rates (will not change DG)

For coupled reactions (1) and (2)

(1) A B ΔG’1

(2) B C ΔG’2

• Standard free-energies for two coupled reactions

are additive: – A C DG ’1+2 = ΔG’1 + ΔG’2

• Equilibrium constants for two coupled reactions is multiplicative:

– A C K'eq 1+2 = (K'eq1)(K'eq2)

Review of Organic Chemistry

• Most reactions in biochemistry are heterolytic processes

– bonding electrons move as pairs (no radicals)

• Nucleophiles react with Electrophiles

– nucleophiles – donate electrons

– electrophiles – seek electrons

• Heterolytic bond breakage often gives rise to transferable groups, such as protons

• Oxidation of reduced fuels often occurs via transfer of electrons and protons to a dedicated redox cofactor

Chemical Reactivity

Most reactions fall within few categories:

• Cleavage and formation of C–C bonds

• Cleavage and formation of polar bonds

• Internal rearrangements

• Eliminations (without cleavage)

• Group transfers (H+, CH3+, PO3

2–)

• Oxidation-Reduction (e– transfers)

Chemistry at Carbon

• Covalent bonds can be broken in two ways

• Homolytic cleavage is very rare

• Heterolytic cleavage is common, but the products are highly unstable and this dictates the chemistry that occurs

• carbanion

• carbocation

Homolytic vs. Heterolytic Cleavage

Common Nucleophiles and Electrophiles in Biochemistry

nucleophiles donate e-

electrophiles seek e-

Carbonyl Groups are Important in Chemical Transformations in Metabolic Pathways

• Carbonyl oxygen is electron withdrawing: electrophilic carbon

• Facilitate carbanion formation

• Delocalizes carbanion negative charge

• Capacity as electron sinks often augmented by metal ion or acid interactions

Examples of Nucleophilic Carbon-Carbon Bond Formation Reactions (common carbonyl chemistry)

related biochemical process

glycolysis

citric acid cycle

fatty acid catabolism

Isomerizations and Eliminations: No Change in Oxidation State

Addition–Elimination Reactions

Group Transfer Reactions

• 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

Phosphoryl Transfer: Nucleophilic Displacement

Nucleophile forms a partial bond to the phosphorous center

giving a pentacovalent intermediate or a pentacoordinated

transition state

Phosphoryl Transfer from ATP

ATP is frequently the donor of the phosphate in the biosynthesis of phosphate esters.

Hydrolysis of ATP is highly favorable under standard conditions

• Better charge separation in products

• Better solvation of products

• More favorable resonance stabilization of products

o DG'° = -30.5 kJ/mol

Actual DG of ATP hydrolysis differs from DG'

• DGp (phosphorylation potential) depends on:

– The standard free energy

– The actual concentrations of reactants and products

• The free-energy change is more favorable if the reactant’s

concentration exceeds its equilibrium concentration

• True reactant and the product are Mg-ATP and Mg-ADP, respectively

• In vivo, energy released by ATP hydrolysis is greater then the

standard free energy change (more negative: ≈ -(50 to 70) kJ/mol)

]MgATP[

]P[]MgADP[ln'

2

i

DD RTGGp

Several phosphorylated compounds have large DG' for hydrolysis

• electrostatic repulsion within the reactant molecule is relieved

• The products are stabilized via resonance, or by more favorable solvation

• The product undergoes further tautomerization

(Phosphoenolpyruvate, 1,3-biphophosphoglycerate, phosphocreatine)

Phosphates: Ranking by the Standard Free Energy of Hydrolysis

Reactions such as PEP + ADP => Pyruvate + ATP are favorable, and can be used to synthesize ATP.

Phosphate can be transferred from compounds with more negative ΔG' to those with less negative ΔG'.

Hydrolysis of Thioesters

• Hydrolysis of thioesters is strongly favorable

– such as acetyl-CoA

• Acetyl-CoA is an important donor of acyl

groups

– Feeds two-carbon units into metabolic

pathways

– Synthesis of fatty acids

• In acyl transfers, molecules other than water

accept the acyl group

Coenzyme A • The function of CoA is to accept and carry acetyl groups • CoA is a reactive thiol group attached to a modified ADP

Molecular Basis for Thioester Reactivity The orbital overlap between the carbonyl group and sulfur is not as good as the resonance overlap between oxygen and the carbonyl group in esters.

ATP

• The energy in the ATP anhydride bonds is not liberated DIRECTLY via hydrolysis

• There is almost always a covalent intermediate

• The phosphoryl-intermediate is converted into product which has lower free energy than the reactants

ATP • When ATP donates a group it is typically via an SN2 mechanism

• The nucleophile is part of the protein/compound that will gain the group

• ATP can also donate a pyro-phosphate or an adenylyl-phosphate

• Adenylylations gain more energy via the cleavage of the pyrophosphate side product

Energy Requirements

• Macromolecular Synthesis

– Construction of proteins and nucleic acids or precursors

• Transport

– Energy is required to transport molecules against concentration gradients

• Motion

– Actin-myosin contractions and cell motility

Shuffling Phosphates: Enzymes

• Nucleoside diphosphate kinase shuffles diphosphates from one class of nucleotide to another

• Adenylate kinase converts 2ADPs into 1 ATP and 1 AMP

• Creatine kinase phosphorylates ADP into ATP using Creatine-monophosphate as a source of Pi

• Polyphosphate Kinases (PPKs) catalyze phosphoryl transfers between polyphosphate and Nucleotides

Oxidation-Reduction Reactions Reduced organic compounds serve as fuels from which electrons can be stripped off during oxidation.

oxid

ation

REDOX (oxidation-reduction reactions)

• Oxidation-reduction reactions are another major source of energy for cells

• Redox is simply electron shuffling

• The forces that accompany the movement of electrons can be optimized to do work

• Oxidation reactions are coupled to reduction reactions

Conjugate Redox Pairs

Fe2+ + Cu2+ Fe3+ + Cu+ electron shuffling split into half reactions: Fe2+ Fe3+ + e- Cu2+ + e- Cu+

electron donor (reductant) e- + electron acceptor (oxidant) reducing agent (reductant): electron donating molecule oxidizing agent (oxidant): electron-accepting molecule

Fe2+ & Fe3+ make up a conjugate redox pair

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–)

• catalyze oxidation reactions

Electron Transfers

• Reducing equivalents refers to the number of electrons transferred in a reaction

• Biological systems use 4 mechanisms to transfer electrons – Directly as electrons (our Fe2+ + Cu2+ example)

– as Hydrogen atoms (one proton + 1 e-)

– as Hydride ion (one proton + 2 e-)

– direct combination with oxygen (oxidation of hydrocarbon to alcohol)

Reduction Potential (E)

• Reduction Potential is a measure of the affinity of the acceptor for electrons

• E’ is the standard reduction potential in V

• More positive E means more affinity for electrons

DE = DE’ + RT ln [electron acceptor] nF [electron donor] R = gas constant (8.315 J/mol*K) T = temperature (K) F = Faraday constant (96,480 J/V*mol) n = number of electrons transferred per molecule

Reduction Potential (E)

• Reduction Potential is a measure of the affinity of the acceptor for electrons

• E0 is the standard reduction potential in V

• More positive E means more affinity for electrons

• We calculate the E via the Nernst Equation

DE = DE’ + 0.026V ln [electron acceptor] n [electron donor] R = gas constant (8.315 J/mol*K) T = 298 K F = Faraday constant (96,480 J/V*mol) n = number of electrons transferred per molecule

Reduction Potential & Free Energy

• Electron acceptor has a higher E' then the donor

∆E' = E'(e- acceptor) – E'(e- donor)

DE'° is positive for energetically favorable reactions

• Reduction potential is related to free energy

DG = -nFDE

DG'° = -nFDE'°

For negative DG need positive DE E(acceptor) > E(donor)

Below are the E’ for the indicated electron carriers: E’ Cyt c1 (Fe3+) + e- Cyt c1 (Fe2+) 0.22 NAD+ + H+ + 2e- NADH -0.32 ½ O2 + 2H+ + 2e- H2O 0.82 Place the electron carriers in order in which they are most likely to act in carrying electrons.

NAD+

Cyt c1

O2

Electron Shuttles

• Oxidation of glucose is used to supply energy for ATP synthesis

• Enzymes act on the glucose to shuffle electrons

• Most redox enzymes use cofactors designed to shuttle electrons

– NADH/NADPH – pyridine nucleotide cofactors

– FMN/FAD – flavin nucleotide cofactors

NAD and NADP are common redox cofactors

• 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

NAD : Nicotinamide adenine dinucleotide NADP: Nicotinamide adenine dinucleotide phosphate

NAD and NADP are common redox cofactors

• NAD+ : usual coenzyme in oxidations

- mitochondrial matrix

• NADPH: usual coenzyme in reductions

- cytosol

functional and spatial specialization

Formation of NADH can be monitored

by UV-spectrophotometry • Measure the change of absorbance at 340 nm

• Very useful signal when studying the kinetics of

NAD-dependent dehydrogenases

Rossmann fold:

• structural motif for binding NAD or NADP in dehydrogenases

• loose association between the dehydrogenase and the coenzyme

• NAD/NADP can readily diffuse from one enzyme to another

• water soluble electron shuttle

Flavin cofactors allow one- or two- electron transfers

• Can participate in a greater diversity of reactions then NAD(P)-linked dehydrogenases

• Also undergo a shift in absorption spectrum upon oxidation

• Permits the use of molecular oxygen as an ultimate electron acceptor

– flavin-dependent oxidases

• Flavin cofactors (FAD & FMN) are tightly bound to proteins

– do not diffuse from enzyme to enzyme

– temporary electron storage for flavoproteins during catalysis

FAD : flavin adenine dinucleotide FMN: flavin mononucleotide

FAD/FMN

Pathway involvement:

• oxidative phosphorylation

• photophosphorylation

• photolyase reactions

Chapter 13: Summary

• 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

– DG depends on Q as well as DG’

– DG dictates whether reactions will occur in the cell

• Unfavorable reactions can be made possible by chemically coupling a highly

favorable reaction to the unfavorable reaction

• Redox reactions commonly involve transfer of electrons from reduced

organic compounds to specialized redox cofactors

– Reduced cofactors can be used in biosynthesis & ATP synthesis