Transcript Enzymes

Enzymes :
Mechanism and Catalysis
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Enzymes DO NOT change the equilibrium constant of a reaction
Enzymes DO NOT alter the amount of energy consumed or liberated in
the reaction (standard free energy change, G°)
Enzymes DO increase the rate of reactions that are otherwise possible
Enzymes DO decrease the activation energy of a reaction (G°‡)
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Enzymes DO increase the rate of reactions that are otherwise possible
Enzymes DO decrease the activation energy of a reaction (G°‡)
The classic way that an enzyme
increases the rate of a bimolecular
reaction is to use binding energy to
simply bring the two reactants in close
proximity. In order for a reaction to
take place between two molecules, the
molecules must first find each other.
This is why the rate of a reaction is
dependent upon the concentrations of
the reactants, since there is a higher
probability that two molecules will
collide at high concentrations. The
enzyme organizes the reaction at the
active site, thereby reducing the cost in
terms of ENTROPY.
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How do enzymes catalyze biochemical reactions?
– involves basic principles of organic chemistry
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What functional groups can be involved in catalysis?
– almost all alpha amino and carboxyl groups are tied up in peptide
bonds
– R groups are involved in catalysis
• asp, glu
• his, lys
• ser, cys, tyr
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catalysis occurs when substrate is immobilized near these residues at
the active site
General Acid-Base Catalysis
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General acid-base catalysis is
involved in a majority of
enzymatic reactions. General
acid–base catalysis needs to be
distinguished from specific
acid–base catalysis.
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In General acid–base catalysis,
the buffer aids in stabilizing the
transition state via donation or
removal of a proton. Therefore,
the rate of the reaction is
dependent on the buffer
concentration, as well as the
appropriate protonation state.
General Base Catalysis I:
Ester Hydrolysis
General Base Catalysis II:
Ester Hydrolysis
The hydrolysis of esters proceeds readily under in the presence of
hydroxide. It is base catalyzed. However, the rate of hydrolysis is
also dependent on imidazole buffer concentration. Imidazole can
accept a proton from water in the transiton state in order to generate
the better nucleophile, hydroxide. It can also re-donate the proton
to the paranitrophenylacetate in order to generate a good leaving
group.
General Acid Catalysis :
Ester Hydrolysis
Electrostatic interactions are much
stronger in organic solvents than in
water due to the dielectric constant of
the medium. The interior of enzymes
have dielectric constants that are similar
to hexane or chloroform
Catalysis by Metal Ions Catalysis I:
Ester Hydrolysis
Metal ions that are bound to the
protein (prosthetic groups or
cofactors) can also aid in
catalysis. In this case, Zinc is
acting as a Lewis acid. It
coordinates to the non-bonding
electrons of the carbonyl,
inducing charge separation, and
making the carbon more
electrophilic, or more susceptible
to nucleophilic attack.
Catalysis by Metal Ions Catalysis
II:
Ester Hydrolysis
Metal ions can also function to make
potential nucleophiles (such as water)
more nucleophilic. For example, the
pka of water drops from 15.7 to 6-7
when it is coordinated to Zinc or
Cobalt. The hydroxide ion is 4 orders
of magnitude more nucleophilic than is
water.
Covalent Catalysis :
Acetoacetate Decarboxylase
O
+
H 3C
Lys
N H3
H 3C
O
O
C
O
CH 3
H 2C
O
hydrolysis
O
CH 3
Lys
N
H
CH 3
B
H 2O
CH 3
Lys
N
H
CH 2
CH 3
O
Lys
O
N
H
CH 2
CO2
BH
• Enzymes physically interact with their substrates to effect
catalysis
• E + S  ES  ES*  EP  E + P
where…
E = enzyme
S = substrate
ES = enzyme/substrate complex
ES* = enzyme/transition state complex
P = product
EP = enzyme/product complex
Enzyme and substrate combine to form a complex
Complex goes through a transition state (ES*)
bound substance is neither substrate nor product
A complex of the enzyme and the product is formed
The enzyme and product separate
All of these steps are governed by equilibria
• Substrates bind to the enzyme’s active site
– pocket in the enzyme
• Substrates bind in active site by
– hydrogen bonding
– hydrophobic interactions
– ionic interactions
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Enzyme/Substrate Interactions
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Lock and key model
substrate (key) fits into a
perfectly shaped space in the
enzyme (lock)
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Induced fit model
substrate fits into a space in the
enzyme, causing the enzyme to
change conformation
change in protein conformation
leads to an exact fit of substrate
with enzyme
Following catalysis, the product(s) no longer fits the active site and is
released
Enzymes and Enzyme Kinetics
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Strain and Distortion model
The binding of the
substrate results in the
distortion of the substrate in
a way that makes the
chemical reaction easier.
Enzyme Kinetics
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The rate of the reaction catalyzed by enzyme E
A+BP
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is defined as
-D[A]
Dt
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-D[B]
Dt
or
D[P]
Dt
Enzyme activity can be assayed in many ways
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or
disappearance of substrate
appearance of product
continuous assay
end point assay
For example, you could measure
– appearance of colored product made from an uncolored substrate
– appearance of a UV absorbent product made from a non-UV-absorbent
substrate
– appearance of radioactive product made from radioactive substrate
Enzymes and Enzyme Kinetics
Higher temperature generally causes more
collisions among the molecules and therefore
increases the rate of a reaction. More collisions
increase the likelihood that substrate will collide
with the active site of the enzyme, thus
increasing the rate of an enzyme-catalyzed
reaction.
Each enzyme has an optimal pH. A change in pH can
alter the ionization of the R groups of the amino acids.
When the charges on the amino acids change, hydrogen
bonding within the protein molecule change and the
molecule changes shape. The new shape may not be
effective.
The diagram shows that pepsin functions best in an acid
environment. This makes sense because pepsin is an
enzyme that is normally found in the stomach where the
pH is low due to the presence of hydrochloric acid.
Trypsin is found in the duodenum, and therefore, its
optimum pH is in the neutral range to match the pH of
the duodenum.
At lower concentrations, the active sites on most of the
enzyme molecules are not filled because there is not
much substrate. Higher concentrations cause more
collisions between the molecules. With more
molecules and collisions, enzymes are more likely to
encounter molecules of reactant.
The maximum velocity of a reaction is reached when
the active sites are almost continuously filled.
Increased substrate concentration after this point will
not increase the rate. Reaction rate therefore increases
as substrate concentration is increased but it levels off.
If there is insufficient enzyme present, the
reaction will not proceed as fast as it otherwise
would because there is not enough enzyme for
all of the reactant molecules. As the amount of
enzyme is increased, the rate of reaction
increases. If there are more enzyme molecules
than are needed, adding additional enzyme will
not increase the rate. Reaction rate therefore
increases as enzyme concentration increases
but then it levels off.
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The velocity (V) of an enzyme-catalyzed reaction is dependent upon
the substrate concentration [S]
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A plot of V vs [S] is often hyperbolic (Michaelis-Menten plot)
Enzymes and Enzyme Kinetics
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The Michaelis-Menten equation describes the kinetic behavior of many
enzymes
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This equation is based upon the following reaction:
SP
k1
k2
E + S  ES  E + P
k-1
V = Vmax [S]
KM + [S]
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the reverse reaction (P  S) is not considered because the equation
describes initial rates when [P] is near zero
Enzymes and Enzyme Kinetics
V = Vmax [S]
KM + [S]
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V is the reaction rate (velocity) at a substrate concentration [S]
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Vmax is the maximum rate that can be observed in the reaction
– substrate is present in excess
– enzyme can be saturated (zero order reaction)
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KM is the Michaelis constant
– a constant that is related to the affinity of the enzyme for the substrate
– units are in terms of concentration
KM = k-1 + k2
k1
Enzymes and Enzyme Kinetics
Enzyme (E) Substrate (S)
Acetylycholine esterase
Carbonic Anhydrase
Carbonic Anhydrase
Catalase
H_2O_2
Fumerase
Fumerate
Fumerase
Malate
Urease
Urea
K_M (in M)
Acetylcholine
CO_2
HCO_30.025
5.0 x 10^{-6}
2.5 x 10^{-5}
0.025
k_{cat} (in s^{-1})
9.5 x 10^{-5} 1.4 x 10^4
0.012
1.0 x 10^6
0.026
4.0 x 10^5
1.0 x 10^7
4.0 x 10^8
800
1.6 x 10^8
900
3.6 x 10^7
1.0 x 10^4
4.0 x 10^5
k_{cat}/K_M (in M^{-1}s^{-1})
1.5 x 10^6
8.3 x 10^7
1.5 x 10^7
Lineweaver-Burk Plot
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KM is also the substrate concentration at which the enzyme operates at
one half of its maximum velocity
if [S] = KM
V = Vmax [S]
2[S]
V = Vmax
2
Enzymes and Enzyme Kinetics
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To determine KM and Vmax, decide on a number of different [S] values,
and measure V at each concentration (hold [E] constant)
Enzymes and Enzyme Kinetics
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Michaelis-Menten plot is not useful for estimating KM and Vmax
it is better to transform the Michaelis-Menten equation to a linear form
– actual values for KM and Vmax determined from graph
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double reciprocal plot or a Lineweaver-Burk plot
Enzymes and Enzyme Kinetics
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By taking the inverse of the Michaelis-Menten equation
V = Vmax [S]
KM + [S]
1 = KM
+ [S]
V Vmax [S]
Vmax [S]
1 = KM . 1 + 1
V Vmax [S] Vmax
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same form as y = mx + b
plot is y vs x
– y is 1/V
– x is 1/[S]
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KM/Vmax is slope
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y intercept is 1/Vmax
x intercept is -1/ KM
Enzymes and Enzyme Kinetics
Enzyme Inhibition
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Certain compounds inhibit enzymes
– decrease the rates of their catalysis
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inhibition can be reversible or irreversible
3 types of reversible inhibitors
– competitive inhibitors
– non-competitive inhibitors
– un-competitive inhibitors
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Irreversible inhibition
– suicide inhibitors
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the various types of inhibitors can be
distinguished by the kinetics of their inhibition
Enzymes and Enzyme Kinetics
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Competitive inhibition
– inhibitor mimics substrate
– fits into active site
• malonate is a competitive inhibitor of
succinate dehydrogenase
Enzymes and Enzyme Kinetics
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Competitive inhibitors can be identified by the kinetics of their inhibition
In the presence of a competitive inhibitor
– KM increases
– Vmax stays the same
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The effects of competitive inhibition can be overcome by increasing [S]
Enzymes and Enzyme Kinetics
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Non-competitive inhibition
– inhibitor binds to a site other than the active site
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Non-competitive inhibitors can be identified by
the kinetics of their inhibition
In the presence of a non-competitive inhibitor
– KM stays the same
– Vmax decreases
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The effects of non-competitive inhibition
cannot be overcome by increasing [S]
Enzymes and Enzyme Kinetics
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Un-competitive inhibition
– inhibitor binds to a site other than the active
site, but only when substrate is bound
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Un-competitive inhibitors can be identified by
the kinetics of their inhibition
In the presence of an un-competitive inhibitor
– KM decreases
– Vmax decreases
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The effects of un-competitive inhibition cannot
be overcome by increasing [S]
Enzymes and Enzyme Kinetics
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Irreversible inhibition
– enzyme is covalently modified after interaction with inhibitor
– derivatized enzyme is no longer a catalyst
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Organofluorophosphates used as insecticides and nerve gases
– irreversible inhibitors of acetylcholinesterase
– form covalent product with active site serine residue
– enzyme no longer functional
Enzymes and Enzyme Kinetics
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When chymotrypsin is treated with DIPF
– only ser 195 reacts is derivatized
– other ser residues are not labeled
– ser 195 is in the enzyme’s active site
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Why is only ser 195 labeled?
– adjacent amino acid residues in active site make ser 195 more reactive
Enzymes and Enzyme Kinetics
The Acyl Enzyme Intermediate
Diisopropylflurophosphate is an inhibitor of chymotrypsin. It diffuses into the active, wherein a
nucleophilic amino acid attacks the phosphate, releasing fluoride anion. This results in a covalent
bond between the nucleophile and the inhibitor. It inhibits the reaction because it blocks entry of
normal substrates.
The enzyme-inhibitor adduct is very stable. Upon hydrolysis of the protein (6 N HCl, 110°C) and
amino acid analysis on the hydrolysate, a novel amino acid was isolated. It was the
diisopropylphosphoryl derivative of serine.
Coenzymes and Prosthetic Groups
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some enzymes employ coenzymes and prosthetic groups at their active
sites
– used for reactions that amino acid R groups can’t perform
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coenzymes
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metals or small organic molecules
not covalently bound to protein
often function as co-substrates
precursors are often vitamins
prosthetic groups
– small organic molecules
– covalently linked to protein
Enzymes and Enzyme Kinetics
Enzyme Regulation :
1. Control of Enzyme Activity Level
A. Noncovalent modifiers cause conformational change between less active and more active states of
the enzyme.
B. Covalent Modification causes interconversion between inactive and active forms of the enzyme.
2. Control the Amount of the Enzyme
A. Isozymes - forms of the enzyme which differ in properties but catalyze the same reaction. For
example, enzyme forms which differ in Vmax and/or Km. The isozymes can be forms found in
different tissues and organs of an animal or for any eukaryotic organism, isozymes can be located in
different parts of the cell. For example, different isozymes of lactate dehydrogenase are found in
muscle and liver. Malate dehydrogenase occurs in different forms in the cytoplasm and the soluble
matrix phase of the mitochondria.
B. Biosynthesis of the enzyme protein can be controlled at the level of the gene via regulation of
transcription (ie synthesis of the enzyme's mRNA). This is more of a molecular biologic type of
regulation and involves molecules which bind to DNA and influence gene expression. This type of
control where the amount of the enzyme is governed can also be done after the mRNA is made, but
this is quite rare. In this mechanism, the mRNA is prevented from being translated and since mRNA is
rather unstable, it is degraded before it is effectively used by the ribosomes to make the protein.
Allosteric Regulation
Control of Enzyme Activity by Non-Covalent Modifiers is usually called allosteric
regulation since the modifier binds to the enzyme at a site other than the active site
but alters the shape of the active site. Allosteric is a word derived from two Greek
words: 'allo' meaning other and 'steric' meaning place or site; so allosteric means
other site and an 'allosteric enzyme' is one with two binding sites - one for the
substrate and one for the allosteric modifier molecule, which is not changed by the
enzyme so it is not a substrate. The molecule binding at the allosteric site is not
called an inhibitor because it does not necessarily have to cause inhibition - so they
are called modifiers. A negative allosteric modifier will cause the enzyme to have
less activity, while a positive allosteric modifier will cause the enzyme to be more
active. In order for allosteric regulation to work, the enzyme must be multimeric (ie.
a dimer, trimer, tetramer etc.). The concept is easily illustrated using a dimer as the
model system, but it applies equally well to higher order multimers such as trimers
and tetramers, etc.
Enzyme can bind two substrates
molecules at different binding sites.
Cooperativity
k1
S+E
k-1
k3
S + C1
k-3
C1
k2
C2
k4
P+E
P+E
or
S
S
E
C1
C2
S
S
P
P
E
E
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The velocity (V) of an enzyme-catalyzed reaction is dependent upon
the substrate concentration [S]
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For allosteric enzymes, a plot of V vs [S] shows a sigmoidal relationship
Enzymes and Enzyme Kinetics
Positive/negative cooperativity
Usually, the binding of the first
S changes the rate at which
the second S binds.
• If the binding rate of the
second S is increased, it’s
called positive cooperativity
• If the binding rate of the
second S is decreased, it’s
called negative cooperativity.
Independent binding sites
k1  2k3  k
2k1  k3  k
2k2  k4
S
S
2k+
E
C1
k-
2k-
C2
S
S
P
P
E
k2e0 s
V 2
Ks
k+
E
Just twice the single
binding rate, as expected
Pseudo-steady assumption
K 2e0 s
c1 
K1K 2  K 2 s  s2
e0 s 2
c1 
2
K1K 2  K 2 s  s
(k2K 2  k 4 s)e0 s
V  k2c1  k 4 c 2 
2
K1K 2  K 2 s  s

Note the quadratic
behaviour
Hill equation
In the limit as the binding of the second S becomes infinitely fast, we get a nice
reduction.
VERY special assumptions, note.
Let k3 , and k1 0, while keeping k1k3 constant.
(k 2K 2  k 4 s)e0 s
Vmax s2
V

2
2
K1K 2  K 2 s  s
K m  s2

Hill equation, with
Hill coefficient of 2.
This equation is used all the time to describe a
cooperative reaction. Mostly use of this equation is
just a heuristic kludge.

http://www.cs.stedwards.edu/chem/Chemistry/
CHEM43/CHEM43/chymotrypsin.mov
Lysozyme
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Lysozyme is a small globular protein composed of 129 amino acids.
It is also an enzyme which hydrolyzes polysaccharide chains,
particularly those found in the peptidoglycan cell wall of bacteria. In
particular, it hydrolyzes the glycosidic bond between C-1 of N-acetyl
muramic acid and C-4 of N-acetyl glucosamine.
It is found in many body fluids, such as tears, and is one of the body’s
defenses against bacteria.
The best studied lysozymes are from hen egg whites and
bacteriophage T4.
Although crystal structures of other proteins had been determined
previously, lysozyme was the first enzyme to have its structure
determined.
Lysozyme Active Site
The X-ray crystal structure of lysozyme has been
determined in the presence of a non-hydrolyzable
substrate analog. This analog binds tightly in the
enzyme active site to form the ES complex, but ES
cannot be efficiently converted to EP. It would not
be possible to determine the X-ray structure in the
presence of the true substrate, because it would be
cleaved during crystal growth and structure
determination.
The active site consists of a crevice or depression
that runs across the surface of the enzyme. Look at
the many hydrogen bonding contacts between the
substrate and enzyme active site that enables the ES
complex to form. There are 6 subsites within the
crevice, each of which is where hydrogen bonding
contacts with the sugars are made. In site D, the
conformation of the sugar is distorted in order to
make the necessary hydrogen bonding contacts.
This distortion raises the energy of the ground
state, bringing the substrate closer to the transition
state for hydrolysis.
General Acid-Base Catalysis in Cleavage by
Lysozyme
At what position does water attack the sugar? When
the lysozyme reaction is run in the presence of H218O,
18O ends up at the C-1 hydroxyl group at site D. This
suggests that water adds at that carbon in the
mechanism. From the X-ray structure, it is known that
the C-1 carbon is located between two carboxylate
residues of the protein (Glu-35 and Asp-52). Asp-52
exists in its ionized form, while Glu-35 is protonated.
Glu can act as a general acid to protonate the leaving
group in the transition state. Asp can function to
stabilize the positively charged intermediate. Glu then
acts as a general base to deprotonate water in the
transition state.
Importance of Strain in Catalysis
Stable Chair
conformation
Distorted boat
conformation
The Serine Proteases
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The serine proteases are a class of enzymes that degrade
proteins in which a serine in the active site plays an
important role in catalysis.
The family includes among many others, Chymotrypsin
and trypsin, which we’ve talked about, and Elastase.
All three enzymes are similar in structure, and they all
have three important conserved residues–a histidine, an
aspartate, and a serine.
Chymotrypsin cleaves after mainly aromatic amino acids,
while trypsin cleaves after basic amino acids. Elastase is
fairly nonspecific, and cleaves after small neutral amino
acids. Notice how their active sites are suited for these
tasks.
Chymotrypsin Mechanism (Step 1)
Chymotrypsin Mechanism (Step 2)
Chymotrypsin Mechanism (Step 3)
Chymotrypsin Mechanism (Step 4)
Chmyotrypsin Mechanism (Step 5)
Chymotrypsin Mechanism (Step 6)
Chymotrypsin Mechanism (Step 7)