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 (25C)
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

G1

(2) B C

G2

Standard free-energies for two coupled reactions

are additive:
A C DG 1+2 = G1 + G2

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 CC bonds
Cleavage and formation of polar bonds
Internal rearrangements
Eliminations (without cleavage)
Group transfers (H+, CH3+, PO32)
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

AdditionElimination 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 reactants
concentration exceeds its equilibrium concentration
True reactant and the product are Mg-ATP and Mg-ADP, respectively
[MgADP ] [Pi ]
DG p DG' RT ln
[MgATP 2 ]

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

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

Several phosphorylated compounds have large

DG' for hydrolysis
(Phosphoenolpyruvate, 1,3-biphophosphoglycerate, phosphocreatine)

electrostatic repulsion within the reactant molecule is relieved

The products are stabilized via resonance, or by more favorable solvation
The product undergoes further tautomerization

Phosphates: Ranking by the Standard Free Energy of

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

Reactions such as
PEP + ADP => Pyruvate + ATP

are favorable, and can be used to

synthesize ATP.

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.

oxidation

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+
Cu2+ + e-

Fe3+ + eCu+

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
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 twoelectron 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