7

The Molecular Basis

Goal To understand how enzymes
accelerate the rates of chemi-chemical reactions, and we saw that many reactions that are essential for
cal reactions.
Objectives
After this chapter, you should be able to
explain:
• why enzymes are necessary for life.
• how enzymes lower ΔG by stabilizing
‡
transition states.
• how enzymes lower ΔG‡ by using
proximity and orientation effects.
• how enzymes enhance the
nucleophilicity and electrophilicity of
reactants.
of Enzymatic Catalysis
In earlier chapters we learned about the factors that influence the rates of
life occur extremely slowly. Consequently, living systems require catalysts
to accelerate the rates of biochemical reactions so that they occur on a
timescale that is consistent with life. We will see numerous examples
of biological catalysts, or enzymes, including DNA polymerase, RNA
polymerase, and kinases in later chapters. Nearly all enzymes are proteins,
but some, as we will see in Chapter 13, are made of RNA instead. We will
explore the arrow pushing mechanisms used by some enzymes (such as the
kinase mechanism), but we first have to establish how enzymes are able to
accelerate reaction rates. In this chapter we will explore the molecular basis
of enzymatic catalysis to understand how and why certain reactions occur
faster with the help of a catalyst.
Enzymes accelerate reactions by lowering ΔG‡
Enzymes do not affect ΔG°rxn; instead, they increase reaction rates by
lowering the activation energy, ΔG‡. As we saw in Chapter 4, an amount
of free energy equal to ΔG‡ must be added to substrates in order to form
the transition state, which then proceeds to form products. The difference
between a fast reaction and one that occurs slowly is simply the value of
ΔG‡; the larger the activation energy barrier, the more energy must be
infused into the substrates before they can become products, and therefore
the slower the reaction. Conversely, reactions with low ΔG‡ values proceed
quickly.
Chapter 7 The Molecular Basis of Enzymatic Catalysis 2

Figure 1 Enzymes bind to their

substrates to catalyze chemical re- substrate
actions
In order for an enzyme to catalyze a chemical
reaction it must first bind to its substrate to
form an enzyme-substrate complex. The
enzyme stabilizes the reaction’s transition substrate
state, making it easier for the bound binding enzyme-substrate
free enzyme
substrate to form the transition state and complex
convert to product. The resulting enzyme-
product complex then dissociates, releasing
free product and regenerating the free
enzyme, which can carry out subsequent
rounds of catalysis.
product catalyzed
release reaction
[TS]‡

product

enzyme-product
complex

Enzymes lower ΔG‡ by allowing reactions to proceed via an alternate

reaction mechanism that has a lower ΔG‡ than the uncatalyzed reaction.
The substrates and products, and therefore their free energy values, are
the same for both the catalyzed and uncatalyzed reactions, thus ΔG°rxn is
the same for both reactions. Since the catalyzed and uncatalyzed reactions
proceed via different mechanisms, however, the structures of the transition
states are not the same for both reactions. Therefore, the free energies of the
transition states are not the same either. The transition state of the catalyzed
reaction is more stable; therefore, the catalyzed reaction has a smaller ΔG‡
and proceeds faster.
At the molecular level, an enzyme-catalyzed reaction unfolds as shown in
Figure 1. First the enzyme randomly encounters the substrate in solution.
Occasionally such an encounter will take place in a manner that allows the
enzyme to bind to the substrate, forming an enzyme-substrate complex.
When bound to the enzyme, the substrate experiences a precisely tailored
environment that facilitates the substrate’s transformation into the transition
state of the reaction. Rather than simply binding to the active site of the
enzyme, the substrate also rearranges itself upon binding to more closely
resemble the transition state. Similarly, the enzyme also undergoes slight
conformational changes to better accommodate the substrate. This creates
more favorable interactions between the substrate and active site and is
called the induced fit model of enzyme catalysis. The energy released by
forming these favorable interactions contributes to lowering the ΔG‡ in
enzyme-catalyzed reactions.
The enzyme-stabilized transition state can then undergo additional
changes to become enzyme-bound product. Since the substrate already
more closely resembles the transition state, less energy is needed to push
Chapter 7 The Molecular Basis of Enzymatic Catalysis 3

Figure 2 Enzymes accelerate reac- [TS]‡

tions by lowering ΔG ‡

Shown are two reaction coordinate

diagrams for a catalyzed reaction (blue line)
and its corresponding uncatalyzed reaction
(red line). The Gibbs free energy difference
∆G‡ uncat
E ([TS]‡)
of the products and reactants is the same
regardless of whether or not the reaction is uncatalyzed rxn
catalyzed; consequently, ΔG°rxn is the same E+S
for both the catalyzed and uncatalyzed ∆G‡ cat
reactions. The catalyzed reaction contains
two local minima corresponding to the G ES
formation of the enzyme-substrate (E S) ∆G° rxn
and enzyme-product (E P) complexes. The
catalyzed reaction proceeds faster because
ΔG‡cat is smaller than ΔG‡uncat. For this to be catalyzed rxn
true, the enzyme must lower the energy of
the transition state more than the energy of E+P
the substrate.

EP
Course of reaction
(reaction coordinate)

the substrate to product. The enzyme-product complex then dissociates

into free product and free enzyme. The released enzyme is then ready to
catalyze the conversion of another molecule of substrate into product. It is
important to note that the enzyme is not changed by the reaction; when the
reaction is complete, the enzyme is in the same state as before the reaction
began.
Figure 2 compares the reaction energy diagram of a catalyzed reaction
to that of an uncatalyzed reaction. As you can see, the catalyst stabilizes
the transition state (i.e., lowers its energy) and reduces ΔG‡ relative to
the uncatalyzed reaction. You can also see as mentioned before that the
energies of the substrates and products are the same for both the catalyzed
and uncatalyzed reactions, thus ΔG°rxn is also the same for both reactions.
Note that some energy is released when the enzyme binds to the substrate;
this loss of energy reflects the formation of favorable interactions between
the substrate and enzyme, thus enabling formation of an enzyme-substrate
complex. Similarly, dissociation of the enzyme-product complex is slightly
unfavorable. Because the enzyme-substrate complex is lower in energy
than the free substrate, you should appreciate that enzymes must stabilize
the transition state more than the substrates in order to decrease ΔG‡. For
example, an enzyme that binds to the substrates and releases an amount
of energy equal to the amount of energy by which the transition state is
stabilized would not lead to a decrease in ΔG‡ because the activation barrier
for the reaction would still be just as large as for the uncatalyzed reaction.
Therefore, an effective catalyst must preferentially stabilize the transition
state.
Chapter 7 The Molecular Basis of Enzymatic Catalysis 4

rate constant
(k)

Probability that Probability that

Reaction = Reactant Molecular Reaction
Rate concentration
x velocity
x cross-section
x molecules collide in x molecules collide with
the right orientation enough energy to react

ΔS‡ ΔH‡
Figure 3 ΔH‡ and ΔS‡ relate to specific factors that affect reaction rate
Shown in the center row are the factors that affect reaction rate. All factors except for reactant concentration are included in the rate
constant. Reaction cross-section and the probability that molecules collide in the right orientation determine ΔS‡, whereas the probability
that molecules collide with enough energy to react determines ΔH‡.

ΔG‡ can be expressed in terms of ΔH‡ and ΔS‡

To understand enzymatic catalysis we need to remind ourselves of the
factors that influence reaction rate. As we have learned, the rate of a
reaction is equal to the concentration of starting material multiplied by the
rate constant (k). The rate constant accounts for all factors that influences
rate except for reactant concentration; therefore, the rate constant accounts
for molecular velocity, reaction cross-section, and the probability that
molecules will collide with enough energy and in the correct orientation
to react (Figure 3). Enzymes increase reaction rate by increasing the value
of the rate constant, which is related to ΔG‡ by the Arrhenius equation that
we saw in Chapter 4: ‡
-∆G

k = A e RT
We can also express ΔG‡ in terms of ΔH‡ and ΔS‡ (ΔG‡ = ΔH‡ - TΔS‡).
The ΔH‡ and ΔS‡ terms represent the changes in enthalpy and entropy,
respectively, between the substrates and transition state (e.g., ΔH‡ = HTS
– Hsubstrates). The ΔS‡ term relates to the reaction cross-section and the
probability that molecules will collide in the correct orientation to react.
Enzymes affect ΔS‡ by reducing the number of conformations the substrate
can adopt. The ΔH‡ term relates to the probability that molecules collide
with enough energy to react. Temperature, as we have discussed already,
affects molecular velocity and the probability that molecules collide with
enough energy to react. As we will see, enzymes can decrease ΔG‡ by
affecting ΔH‡ and ΔS‡.

Enzymes use proximity and orientation effects to increase reaction rate

To accelerate chemical reactions, enzymes make use of a variety of molecular
strategies, often with a remarkable effectiveness that is unmatched by
analogous catalysts developed in the laboratory. A major strategy, called the
Chapter 7 The Molecular Basis of Enzymatic Catalysis 5

Figure 4 The proximity effect in-

creases collision frequency
The top row shows an intermolecular
reaction in which two reactants, A and B, A + B A B
form a new bond to each other. The bottom
row shows an equivalent intramolecular increasing rxn rate,
reaction in which A and B are already more favorable ΔS‡
connected. Intramolecular reactions are A B A B
usually faster than intermolecular reactions
because the reactants are held together in
space (proximity effect) and collide more
frequently as a result.

proximity effect, is to organize the substrates within the active site of the
enzyme such that the reactants are much closer together than they would be
in a typical solution. Enhancing the proximity of reactants increases their
collision frequency, thus causing the reaction to proceed at a faster rate. The
proximity effect effectively increases the local concentration of substrate
(recall that rate is proportional to substrate concentration).
As an example of the proximity effect in catalysis, consider the rates of the
two hypothetical reactions shown in Figure 4. The reaction at the top relies
on the random collision between the two substrates to bring A and B close
enough to react. In contrast, it is much more likely for A and B to encounter
each other in the reaction at the bottom when they are already tethered
together.
In addition to proximity effect, a related but distinct strategy used by
enzymes to accelerate chemical reactions is to orient substrates into a
maximally reactive conformation; this is known as the orientation effect.
Simply confining two substrates close to one another does not guarantee a
faster reaction rate because in order for two substrates to react they usually
must achieve a specific relative orientation. This is similar to you finding
your friend in a dining hall. Not only do you need to be in the same area
at the same time, but you also need to be facing each other. In the example
in Figure 5, it is not sufficient to simply tether A and B together; instead, a
requirement of their reaction is to orient A and B properly. Many enzymes
catalyze reactions not only by holding substrates close together, but also by
forcing the substrates into an optimal orientation to lower the activation
energy needed to reach the transition state.
Proximity and orientation effects lower the entropic barrier to forming the
transition state (ΔS‡) because they pre-organize the substrates so that they
lose less entropy during the formation of the transition state than the free
substrates would. In other words, when enzymes bind to substrates they
often reduce the entropy of the substrates by constraining them into reactive
conformations, thus the favorability of binding between the enzyme and
the substrates effectively “pre-pays” for the loss of entropy that is required
to form the transition state.
We can quantify the proximity and orientation effects using the effective
concentration of the reactants in the reactions. The effective concentration
is defined as the ratio of the rate constant for the intramolecular reaction
(with units of s-1) divided by the rate constant for the intermolecular
Chapter 7 The Molecular Basis of Enzymatic Catalysis 6

B
B
A A B
A

B
increasing rxn rate,
A B more favorable ΔS‡
A

A B

reactive conformation

Figure 5 Substrates that are restrained into a reactive conformation react more rapidly
Shown are three hypothetical substrates (top, middle, and bottom rows) in which the reactive regions, A and B, are connected by covalent
bonds. The reactive conformation in which A and B are close together is drawn in the right-most column for each substrate. The substrate
on the top row is the least constrained of the three and can adopt a large number of conformations; consequently, it must lose a large
amount of entropy in order to adopt the reactive conformation, making ΔS‡ less favorable. In contrast, the substrate on the bottom row is
already constrained into a reactive conformation; consequently, no loss of entropy is needed to adopt the reactive conformation, making
ΔS‡ more favorable.

reaction (with units of s-1M-1). Effective concentration has units of molarity

(M) and is a measure of the concentration of a reactant you would have to
have in an intermolecular reaction to achieve the same rate as that in the
intramolecular reaction with a substrate concentration of 1M. In the example
shown in Figure 6, the rate constant for the intermolecular reaction (top) is
kinter = 4 x 10-6 s-1M-1; in contrast, the rate constant of the intramolecular
reaction (bottom) is kintra = 0.8 s-1. The effective concentration of A and
B in the bottom case is kintra/kinter, or 200,000 M! In other words, A and
B when tethered in close proximity and in the proper orientation by an
enzyme react at the same rate as that of the untethered reaction when A
or B is an impossibly high 200,000 M. Note that effective concentration
takes into account both proximity and orientation effects; therefore, if the
reactants were tethered close to each other but held firmly in the incorrect
orientation for the reaction, the effective concentration would not be high.
Enzymes use orientation and proximity effects, often with dramatic results,
by confining reactants in the proper orientation within the relatively small
active site of the enzyme.

Enzymes use acid and base catalysis to affect ΔH‡

In addition to proximity and orientation affects, which affect ΔS‡, enzymes
also catalyze reactions by providing proton donors (acids) and proton
acceptors (bases) at precise locations in the active site. These strategies
Chapter 7
Figure 6 Effective concentration
measures the rate enhancement
of an intramolecular reaction rel-
ative to a corresponding intermo-
lecular reaction
(A) Shown are two hypothetical reactions
between reactants A and B. The top row
shows an intermolecular reaction while the
bottom row shows an otherwise equivalent
intramolecular reaction. The rate constants
of both reactions, kinter and kintra, are
indicated. (B) Effective concentration
(Ceff ) equals the ratio of the intramolecular
rate constant over the intermolecular
rate constant. In this example Ceff equals
200,000 M, meaning that the reactants in
the intermolecular reaction would have to
be present at 200,000 M in order to match
the rate of the intramolecular reaction
when its reactants are present at 1 M.
The Molecular Basis of Enzymatic Catalysis 7
(A)
kinter = 4 x 10-6 M-1s-1
A + B A B
kintra = 0.8 s-1
A B A B
(B) kintra 0.8 s-1
Ceff = = = 200,000 M
kinter 4 x 10 M s
-6 -1 -1
largely affect ΔH‡, accelerating the rate of breakage and formation of
bonds. In the laboratory chemists can accelerate many reactions by adding
acid or base to lower or raise the pH of the reaction solution. These pH
changes typically accelerate reactions by altering the nucleophilicity or
electrophilicity of the reactants.
As its name implies, nucleophilicity describes the effectiveness of a
nucleophile and is a measure of how quickly an atom is able to form a
covalent bond with an electron-deficient atom. Highly nucleophilic atoms
are able to form bonds faster than weak nucleophiles. Nucleophilicity is a
kinetic parameter because it reflects the rate of bond formation rather than
the amount of energy released as a result of bond formation.
Nucleophiles can vary widely in their nucleophilicity, and as you would
expect, the best nucleophiles tend to be atoms with an excess of negative
charge. As a general rule of thumb, when comparing two atoms of similar
size (such as among N, O, or F), the atom that is the strongest base (whose
conjugate acid has the higher pKa) is the more nucleophilic atom. Therefore,
a hydroxide anion (OH-), whose conjugate acid (H2O) has a pKa of 15.5, is a
much better nucleophile than water, whose conjugate acid (H3O+) has a pKa
of -1.5 (Figure 7). Likewise, ammonia (NH3), whose conjugate acid (NH4+)
has a pKa of 10, is a better nucleophile than water.
Conversely, electrophilicity describes the effectiveness of an electrophile
and measures how quickly an electron-deficient atom is able to form
a covalent bond with an incoming nucleophile. Like nucleophilicity,
electrophilicity is a kinetic parameter. As you might expect, those atoms
attached to groups that can most readily accept electrons (including groups
made of electronegative atoms) are the most electrophilic and make the
best electrophiles. In the chemical reactions involved in life, electrophilic
atoms are most often carbon or phosphorus atoms that are doubly bonded
to oxygen atoms (Figure 8). If you take organic chemistry you will learn in
much more detail the factors that determine electrophilicity, but for our
purposes, we will focus on C=O and P=O groups as electrophiles.
Chapter 7 The Molecular Basis of Enzymatic Catalysis 8

Figure 7 Stronger bases are often Stronger base,

better nucleophiles better nucleophile
For atoms of similar size, stronger bases
are generally more nucleophilic. Shown
are three bases—water, ammonia, and H
hydroxide—arranged from left to right Base O
H H N H O
in order of increasing basicity and H H
nucleophilicity. The conjugate acid of each
base is also shown, along with the conjugate
acid’s pKa. Stronger bases have conjugate H H
Conjugate O
acids with higher pKa values.
Acid O H N H H H
H H H

pKa -1.5 9.25 15.5

Figure 8 Carbonyl and phosphate O

groups are common electrophiles O
O P O
in living systems C
Shown are the structures of a carbonyl and
O
phosphate group. The electrophilic atom is
shown in red in both structures. carbonyl phosphate

The protonation or deprotonation of a group can profoundly change its

nucleophilicity or electrophilicity. As we have already seen in the case
of water, removing a proton from a group typically increases its electron
richness, and therefore its nucleophilicity. Conversely, adding a proton to
a group typically makes that group more readily accept electrons, thereby
increasing its electrophilicity. For example, the protonation of a carbonyl
oxygen (Figure 9) results in a dramatic increase in the ability of the carbonyl
carbon to accept an incoming nucleophile. This is because the protonation
of the oxygen causes oxygen to become more positive because it loses one
of its lone pairs and is not sharing those electrons with hydrogen. The
positive charge on the oxygen causes the carbon-oxygen double bond to be
even more polar than usual, with electrons in the bond being attracted to
the positively charged oxygen. Since these electrons are more attracted to
the protonated oxygen, the carbon atom becomes more positive and more
electron-deficient, making it more electrophilic.

Figure 9 Protonation state affects base

nucleophilicity and electrophilici- -H+ more
ty O H O electron-
H H rich
Deprotonation by a base (top) increases an better nucleophile
atom’s nucleophilicity by making it more
electron-rich. Conversely, protonation by
an acid (bottom) increases a functional
group’s electrophilicity. In this example more
protonation of the carbonyl oxygen makes acid H electron-

the C-O double bond more polar (electrons

O +H+ O deficient

are more attracted to the positively charged C C

oxygen), which in turn makes the carbon
atom more electron-deficient. better electrophile
Chapter 7 The Molecular Basis of Enzymatic Catalysis 9

δ−
O O
Weaker nucleophile:
C C N slower reaction,
N H
H higher ΔG‡
O
H δ+ H
O
H H

δ−
O
O Stronger nucleophile:
C C N faster reaction,
N H
H − lower ΔG‡
Oδ
H
Water + Base O
H
Figure 10 Base catalysis accelerates amide bond hydrolysis by increasing the nucleophilicity of water
Shown is the example of amide bond hydrolysis in neutral water (top) and in a basic solution (bottom). The presence of base leads to the
deprotonation of water, which increases its nucleophilicity. The stronger nucleophile leads to a faster reaction because the accompanying
transition state that is lower in energy than the transition state in which water is used as a nucleophile.

As an example, let us consider amide bond hydrolysis. You can now

appreciate that neutral water is only weakly nucleophilic, whereas
deprotonated water (the hydroxide anion) is a more effective nucleophile.
A chemist might speed up the rate of amide bond hydrolysis in a test tube
by adding base in order to deprotonate water and thereby convert it into a
better nucleophile (Figure 10). Although enzymes in general cannot change
the pH of an entire cell, they can use acid catalysis and base catalysis by
positioning acidic or basic groups at just the right locations to protonate or
deprotonate particular groups within a substrate. Acid and base catalysis
lowers ΔH‡, thus decreasing ΔG‡ as well. Most commonly these acids and
bases are the side chains of acidic and basic amino acids.
Even though we can draw reasonable transition state structures for the
cases presented in Figure 10, it is not obvious simply from inspecting the
structures of these transition states which would have a smaller ΔG‡ value.
Instead, you should reason that the hydroxide anion is a better nucleophile
than water; therefore, the hydroxide anion will more rapidly form a bond
to the electrophile than water, and thus the faster rate will correspond to a
lower ΔG‡ value.

Proteases catalyze peptide bond cleavage

As an example, proteases are enzymes that catalyze the cleavage of peptide
bonds (Figure 11), which is chemically the same reaction as the amide
hydrolysis example that we just examined. We saw the peptide bond cleavage
reaction in Chapter 4 when we examined its arrow pushing mechanism.
We also saw the active site of a protease called chymotrypsin in Chapter 5
as an example of how amino acids can function cooperatively. Your body
Chapter 7 The Molecular Basis of Enzymatic Catalysis 10

O R O R
H H
N + O N + H
N H H OH N
H
O R O O R H O
protein substrate carboxylic acid amine
product product

Figure 11 Proteases catalyze peptide bond hydrolysis

Shown is the chemical reaction of the peptide bond hydrolysis reaction that is catalyzed by proteases. This reaction is thermodynamically
favorable, but it proceeds slowly without a catalyst. Proteases greatly accelerate the reaction’s rate.

contains hundreds of different proteases. Some of the best studied examples

of proteases are those involved in digestion, such as pepsin and trypsin.
These proteases play key roles in allowing your body to make use of protein
nutrients by breaking proteins down into smaller pieces. These protease
digestion products are eventually processed into amino acids that can be
used for many functions in the cell, including the synthesis of new proteins
through the process of translation.
The reaction catalyzed by proteases, peptide bond cleavage, is highly
favorable; therefore, its ΔG°rxn is negative. Although peptide bond hydrolysis
is favorable, it does not proceed rapidly without a catalyst. In fact, this
reaction is so slow that the half-life of a peptide bond in neutral water is on
the timescale of years. Proteases, however, increase the rate of peptide bond
hydrolysis by over a billion-fold, accelerating peptide bond hydrolysis to a
timescale that is consistent with biological processes.

Summary
Many biological reactions do not occur on a timescale that is consistent
with life. Living systems address this problem by using enzymes, which
accelerate otherwise slow chemical reactions by lowering ΔG‡. In contrast,
enzymes do not affect thermodynamics and do not change ΔG°rxn.
Enzymes catalyze reactions by first binding to free substrate to form an
enzyme-substrate complex. In this complex, the enzyme stabilizes the
reaction’s transition state, thereby lowering the overall energy and allowing
substrate to convert more rapidly to product. The resulting enzyme-product
complex then dissociates, releasing free product and regenerating the free
enzyme, which can take part in subsequent rounds of catalysis. In order to
decrease ΔG‡, the enzyme must preferentially stabilize the transition state
relative to the substrate.
One strategy used by enzymes to lower ΔG‡ is the proximity effect, in
which the enzyme constrains the substrates by holding reactive atoms in
close proximity to each other, thereby increasing collision frequency and
decreasing ΔS‡. Similarly, enzymes use the orientation effect, in which
the enzyme constrains the substrates into specific reactive conformations,
thereby increasing the probability that reactants will collide in the correct
orientation to react, thus decreasing ΔS‡ as well.
Chapter 7 The Molecular Basis of Enzymatic Catalysis 11
Enzymes also catalyze reactions using acid and base catalysis by providing
proton donors (acids) and proton acceptors (bases) at precise locations
in the active site. These strategies largely affect ΔH‡ and accelerate the
rate of bond breakage and formation. In base catalysis, a base is used to
deprotonate an atom, thereby giving it additional electron density and
making it more nucleophilic. In acid catalysis, on the other hand, an acid
is used to protonate an atom, thereby making it more positive, typically
increasing the electrophilicity of atoms bonded to it. Acid/base catalysis
can also accelerate the making/breaking of bonds by neutralizing high
energy charges that develop during the course of a reaction. As we will see
later, an example of this can be found in proteases, which are enzymes that
accelerate the hydrolysis of peptide bonds.

Practice problems

1. Shown below is a reaction catalyzed by carbonic anhydrase, an enzyme present in red blood cells. The
active site of the enzyme shown below, facilitates the generation of OH-. The three histidine residues
on the left bind an important Zn2+ ion. Describe the different ways this Zn2+ ion catalyzes the reaction
mechanism.
Chapter 7 The Molecular Basis of Enzymatic Catalysis 12

Solutions to practice problems

Question 1:
• Proximity and orientation effects: The Zn2+ ion positions the water molecule correctly oriented in the
active site of the enzyme in close proximity of the histidine residue, which increases both the collision
frequency of the reactants and the probability that reactants collide in the right orientation.
• Increasing electrophilicity: The Zn2+ ion places positive charge next to the oxygen of H2O, which makes
the hydrogen atom a better electrophile.
• Stabilizing transition state: The positive charge of the Zn2+ ion also stabilizes the formation of the nega-
tive charge on the oxygen of H2O and thus stabilizes the transition state, lowering the activation energy.