Lecture 8 – Enzyme
Energetics
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Last Lecture
We talked about protein conformational
change, signal cascades, phosphorylation,
and ATP. We shall review these things
even more in depth today…
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In this lecture
• Physics!!
– Energy
– The laws of
thermodynamics
– Free energy
• Metabolism
– The role of ATP
• Enzymes
– Enzyme inhibitors and
regulators
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What is energy?
• The ability to do work
• Comes in two main forms
– Potential energy
• Example: Chemical Energy
• Example: Gravitational Energy
– Kinetic energy
• Example: Thermal energy (heat)
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A diver has more potential
energy on the platform
than in the water.
Climbing up converts the kinetic
energy of muscle movement
to potential energy.
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Diving converts
potential energy to
kinetic energy.
A diver has less potential
energy in the water
than on the platform.
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The behavior of energy is governed
by the laws of thermodynamics
• 1st law of thermodynamics: energy cannot be
created or destroyed
• 2nd law of thermodynamics: energy transfer or
transformation is never 100% efficient. Part of the
energy is lost
– Energy transfer increases the entropy (disorder) of
the universe
• 3rd law of thermodynamics: the entropy of a
perfect crystal at a temperature of absolute zero
is zero
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Entropy
• The universe’s tendency towards disorder
• A system (such as an organism) requires
energy input in order to not succumb to
entropy
• What happens when entropy is at maximum
for the entire universe?
– Heat death of the universe
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What does entropy have to do
with life?
• Living cells unavoidably convert organized
forms of energy to heat. We increase the
entropy of the universe.
• Energy flows into an ecosystem in the form of
light and exits in the form of heat
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Does life violate the second law
of thermodynamics?
Life is highly organized, which seems the opposite of what entropy says should happen
• Single organisms are open systems
– Energy and mass freely flow in and out
• Earth is a (mostly) closed system
– Massive amounts of energy flows in from the sun;
relatively little energy leaves
– That surplus of energy is used to build up
complexity
In the long-term (billions of years) life and the Earth will eventually succumb to
entropy. But for now, we’re okay
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Earth’s energy budget
• About 3, 850,000 exajoules of sunlight energy
is absorbed the Earth yearly
1027 x
energy
difference
– Plants capture about 3,000 exajoules/year
– Total energy use in the U.S. is ~94 exajoules
– Energy released in the 2011 Japanese earthquake
and tsunami is ~1.1 exajoules
– The kinetic energy of a flying mosquito is about
1/160th of a nanojoule
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Measuring energy
• If we have a way to measure change in energy,
we have a way to measure how likely a
chemical reaction will spontaneously take
place
• A living system’s free energy (∆G) is energy
that can do work when temperature and
pressure are uniform, as in a living cell
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Free energy as an indication of
stability
• Only processes with a negative ∆G are
spontaneous
• Spontaneous processes can be harnessed to
perform work
• A more stable system has the lowest amount
of free energy
– Less probability of a spontaneous chemical
reaction coming around and changing things
– Equilibrium is a state of maximum stability
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Figure 8.5
• More free energy (higher G)
• Less stable
• Greater work capacity
In a spontaneous change
• The free energy of the system
decreases (G  0)
• The system becomes more
stable
• The released free energy can
be harnessed to do work
• Less free energy (lower G)
• More stable
• Less work capacity
(a) Gravitational motion
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(b) Diffusion
(c) Chemical reaction
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Entropy and biochemical reactions
• Entropy determines if biochemical reactions
will spontaneously take place
– Entropy favors a reaction that increases disorder
– Diffusion is a spontaneous process because it
increases entropy
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Entropy and chemical reactions
• An exergonic reaction proceeds with a net release
of free energy and is spontaneous
• An endergonic reaction absorbs free energy from
its surroundings and is not spontaneous
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Thermodynamics and biology research
Energetics of the HIV gp120-CD4 binding reaction
“The binding thermodynamics were of unexpected magnitude;
changes in enthalpy, entropy, and heat capacity greatly exceeded
those described for typical protein-protein interactions. These
unusual thermodynamic properties were observed with both intact
gp120 and a deglycosylated and truncated form of gp120 protein
that lacked hypervariable loops V1, V2, and V3 and segments of its N
and C termini. Together with previous crystallographic studies, the
large changes in heat capacity and entropy reveal that extensive
structural rearrangements occur within the core of gp120 upon
CD4 binding. CD spectral studies and slow kinetics of binding
support this conclusion. These results indicate considerable
conformational flexibility within gp120, which may relate to viral
mechanisms for triggering infection and disguising conserved
receptor-binding sites from the immune system.”
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The role of ATP
• ATP powers cellular work by coupling
exergonic reactions to endergonic reactions
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What ATP powers
• A cell does three main kinds of work
– Chemical
– Transport
– Mechanical
• To do work, cells manage energy resources by
energy coupling, the use of an exergonic process
to drive an endergonic one
• Most energy coupling in cells is mediated by ATP
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Figure 8.10
Transport protein
Solute
ATP
ADP
P
Pi
Pi
Solute transported
(a) Transport work: ATP phosphorylates transport proteins.
Cytoskeletal track
Vesicle
ATP
ADP
ATP
Motor protein
Pi
Protein and
vesicle moved
(b) Mechanical work: ATP binds noncovalently to motor
proteins and then is hydrolyzed.
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Coupling spontaneous processes to
nonspontaneous ones
• A cotransport protein can couple “downhill”
passive diffusion to a second “uphill” active
transport of a different substance
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ADP to ATP regeneration
• ATP is hydrolyzed into ADP in biochemical
reactions, but then what happens?
• The cell recycles, turning ADP back into ATP
ATP
Energy from
catabolism (exergonic,
energy-releasing
processes)
H2O
ADP
Pi
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Energy for cellular
work (endergonic,
energy-consuming
processes)
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Metabolic Reactions
• Metabolism is the totality of an organism’s
chemical reactions
– A metabolic pathway begins with a specific molecule
and ends with a product
– Each step is catalyzed by a specific enzyme
Enzyme 2
Enzyme 1
A
Reaction 1
Starting
molecule
B
Reaction 2
Similar to a signal
transduction
pathway
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Enzyme 3
C
Reaction 3
D
Product
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Types of metabolic reactions
• Catabolic pathways release energy by
breaking down complex molecules into
simpler compounds
• Anabolic pathways consume energy to
build complex molecules from simpler ones
– The synthesis of a polypeptide from amino acid
monomers is an anabolic pathway
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The activation energy barrier
• Every chemical reaction between molecules involves
bond breaking and bond forming
• The initial energy needed to start a chemical reaction
is called the free energy of activation, or activation
energy (EA)
• Thermal energy from the surroundings often supplies
the activation energy
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Figure 8.12
A
B
C
D
Free energy
Transition state
A
B
C
D
EA
Reactants
A
B
G  O
C
D
Products
Progress of the reaction
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How enzymes lower EA
• Enzymes catalyze reactions by providing another
lower-energy way for the reaction to take place
• Enzymes do not affect the change in free energy
(∆G); instead, they hasten reactions that would occur
eventually
Biology definition: Enzymes lower Ea
Chemistry definition: Enzymes
provide another lower-energy
pathway
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Figure 8.13
Free energy
Course of
reaction
without
enzyme
EA
without
enzyme
EA with
enzyme
is lower
Reactants
G is unaffected
by enzyme
Course of
reaction
with enzyme
Products
Progress of the reaction
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Enzyme vocabulary
• The reactant that an enzyme acts on is called the
enzyme’s substrate
• The enzyme binds to its substrate, forming an
enzyme-substrate complex
• The active site is the region on the enzyme where
the substrate binds
• Induced fit of a substrate brings chemical groups of
the active site into positions that enhance their
ability to catalyze the reaction
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The suffix “-ase”
denotes an
enzyme
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Figure 8.15-1
1 Substrates enter active site.
2 Substrates are held
in active site by weak
interactions.
Substrates
Enzyme-substrate
complex
Active
site
Enzyme
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Figure 8.15-2
1 Substrates enter active site.
2 Substrates are held
in active site by weak
interactions.
Substrates
Enzyme-substrate
complex
3 Active site can
lower EA and speed
up a reaction.
Active
site
Enzyme
4 Substrates are
converted to
products.
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Figure 8.15-3
1 Substrates enter active site.
2 Substrates are held
in active site by weak
interactions.
Substrates
Enzyme-substrate
complex
3 Active site can
lower EA and speed
up a reaction.
6 Active
site is
available
for two new
substrate
molecules.
Enzyme
5 Products are
released.
4 Substrates are
converted to
products.
Products
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The enzymatic process
• The enzyme binds its substrate on the
active site
– An enzyme-substrate complex forms
• Induced fit brings reactive functional groups
of the enzyme into contact with the substrate
• The enzyme breaks and reforms the chemical
bonds of its substrate
• The enzyme releases the products
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Enzyme reactions: looking closer
Enzymes work similarly to proteins in their binding
specificities
Only certain types of enzymes will bind certain
substrates – lock and key
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Figure 8.14
Substrate
Active site
Enzyme
(a)
Enzyme-substrate
complex
(b)
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Some examples of enzymatic
reactions
• The enzyme sucrase breaks down the sucrose
disaccharide into its monomers glucose and
fructose through hydrolysis
Sucrase
Glucose
(C6H12O6)
Sucrose
(C12H22O11)
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Fructose
(C6H12O6)
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Enzymes in biochemical pathways
3, phosphoglycerate is a
product of the
phosphoglycerate kinase
reaction, but a reactant
for the phosphoglycerate
mutase reaction
Blue = enzyme
Black = reactant/product
Green = energy used
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What affects enzymatic activity?
• Protein activity is affected by:
– Temperature
– pH
– Salt concentrations
Enzymes are proteins, and are affected by the same things!
All proteins have an optimal temperature and
pH under which they operate
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Humans and
bacteria have
different sets
of proteins
that operate
at difference
optimal
temperatures
Same with
pH
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What else affects enzyme activity?
• Cofactors/coenzymes
• Competitive/noncompetitive inhibitors
• Allosteric regulators
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What else affects enzyme activity?
• Cofactors and coenzymes can also enhance
or inhibit enzyme activity
• Cofactors are nonprotein enzyme helpers
– Cofactors may be inorganic (such as a metal in
ionic form) or organic
• An organic cofactor is often called a
coenzyme
– Coenzymes include vitamins
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Cofactors and coenzymes
• Metal ion cofactors
– The trace elements required in our diet are often
metal ion cofactors
– Zinc in alcohol dehydrogenase
– Magnesium in glucose-6-phosphatase
• Vitamins
– Folic acid
– Vitamin C
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Enzyme inhibitors
• Inhibitors are special molecules that slow
down or halt enzymes
• Competitive inhibitors bind to the active
site of an enzyme, competing with the
substrate
• Noncompetitive inhibitors bind to another
part of an enzyme, causing the enzyme to
change shape and making the active site less
effective
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Figure 8.17
(a) Normal binding
(b) Competitive inhibition
Substrate
Active
site
(c) Noncompetitive
inhibition
Competitive
inhibitor
Enzyme
Noncompetitive
inhibitor
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Reversible vs. irreversible
inhibitors
• Reversible inhibitors can detach themselves
from the enzyme, allowing it to become active
again
• Irreversible inhibitors either covalently modify
or bind to the enzyme and permanently
disable it
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Examples of inhibitors
• Protease inhibitor – a competitive reversible
inhibitor of the HIV protein protease
Ritonavir, an HIV protease inhibitor
Ritonavir bound to HIV protease in the active site
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Examples of inhibitors II
• DFP – diisopropyl fluorophosphate – is a
potent neurotoxin
– Inhibits acetylcholinesterase, which breaks down
the neurotransmitter acetylcholine
– If acetylcholine is not broken down, it accumulates
and nerve impulses cannot be stopped
– Prolonged muscle contraction
• Irreversible noncompetitive inhibitor
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Allosteric Regulators
• Allosteric regulation may either inhibit or
stimulate an enzyme’s activity
• Allosteric regulation occurs when a regulatory
molecule binds to a protein at one site and
affects the protein’s function at another site
“Allo” = “at
a distance”
Noncompetitive inhibitors
and allosteric regulators can
have the same mechanism of
action
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The whole picture
Human glyoxalase I
Two zinc ion cofactors are shown in purple
A competitive inhibitor called S-hexylglutathione is shown as a
space-filling model in green, blue and red. It is in the active site.
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Inhibitors and Allosteric
Regulators in Drugs
• How a drug, protein or enzyme works is
called its mechanism of action
• Starting with a specific biological target then
creating a molecule designed to affect it is
called rational drug design
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SSRIs
• Selective serotonin reuptake inhibitors
– Zoloft, Celexa, Prozac
– Serotonin is used as a neurotransmitter, a
chemical signal used in cell communication
– Low levels of serotonin is believed to cause
depression
• Used to treat depression
• The first class of drugs to use rational drug
design
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The normal nerve cell
• A nerve cell releases serotonin
as a local signal
• Serotonin binds to serotonin
receptors on an adjacent nerve
cell and the signal is sent
• The serotonin is then released
from the adjacent nerve cell
• The original nerve cell reuptakes
the leftover serotonin back into
the cell through a transporter
protein
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SSRI mechanism of action
• SSRIs slow the reuptake of serotonin by the
original nerve cell
– An SSRI is a competitive inhibitor of the
serotonin reuptake transporter. Serotonin is the
normal ligand
• Serotonin repeatedly binds the receiving cell,
causing the same chemical signal to be sent
repeatedly
Most serotonin
receptors are
GPCRs
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Serotonin
Celexa/Citalopram
Zoloft/Sertraline
http://www.rcsb.org/pdb/explore/jmol.do?struc
tureId=3GWU&bionumber=1
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Useful Links
• http://simple.wikipedia.org/wiki/Thermodynam
ic_entropy - a simple explanation of entropy
• http://bcs.whfreeman.com/thelifewire/content/
chp06/0602002.html - interactive animation of
allosteric regulation
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Vocabulary
• 1st law of
thermodynamics
• 2nd law of
thermodynamics
• Entropy
• Energy of activation
• Energy coupling
• Endergonic, exergonic
reactions
• Metabolism
• Catabolic, anabolic
reactions
• Substrate
• Active site
• Competitive,
noncompetitive
inhibitors
• Allosteric Regulators
• Mechanism of action
• Rational drug design
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