HOW ENZYMES WORK

Enzymes and catalysis

Transition State Theory, Activation

Energy (Ea), & The Reaction
Coordinate
Transition state theory was developed to describe
chemical reactions by applying thermodynamic
equilibrium concepts.
 CATALYTIC MECHANISMS
Enzymes are effective as catalysts due to:

1. Their ability to rearrange covalent

bonds using their various amino acid
side chains, metal ions and coenzymes.

2. Their ability to specifically bind the

substrate molecule in an enzyme-
substrate complex and to use
noncovalent interactions for binding to
significantly lower the free energy.

Once a substrate is bound to an
enzyme, properly positioned catalytic
functional groups aid in the cleavage
and formation of bonds by a variety of
mechanisms, including general acid-
base catalysis, covalent catalysis, and
metal ion catalysis.
• Reactive groups at the active
site catalyze reactions by:
– donating or withdrawing
electrons
– stabilizing or generating free
radical intermediates
– forming temporary covalent
bonds (a transition state
intermediate)
• There is high degree of
specificity for the reaction
catalyzed
 Enzyme Specificity: Types of specifity
Enzymes selectively recognize proper substrates over other
molecules
1. Absolute specifity: enzymes act only on one particular substrate (exp:
Urease specific for urea only)

2. Bond Specificity: Most of the proteolytic enzymes are showing group

(bond) specificity. For example, trypsin can hydrolyse peptide bonds
formed by carboxyl groups of arginine or lysine residues in any proteins

3. Group Specificity: One enzyme can catalyse the same reaction on a

group of structurally similar compounds, e.g. hexokinase can catalyse
phosphorylation of glucose, galactose and mannose

4. Stereospecificity: the enzyme is specific not only to the substrate but

also to its optical configuration
 • L-amino acid oxidase----acts only on L-amino
acids
• D-amino acid oxidase----acts only on D-amino
acids

Enzymes can distinguish between isomers

Enzymes Affect Reaction Rates, Not Equilibrium
of a Reaction

❖ The function of an enzyme is to increase the rate of a

reaction. They do not affect reaction equilibria.

❖ Under biologically relevant conditions, uncatalyzed

reactions tend to be slow. They create an environment to
make the reaction energetically more favorable.
This is the first important rule to understand any
kind of catalysis catalysts do not change the overall
energy of a reaction.

The second important point about catalyzed reactions

–catalysts work by lowering activation energies of
reactions and thus molecules more easily reach the
energy necessary to get to the point where the
reaction occurs.
Activation energy is defined as the energy required to convert all molecules of
a reacting substance from the ground state to the transition state.
 Reaction coordinate diagram comparing enzyme-catalyzed and
uncatalyzed reactions

A catalyst functions by
lowering the activation
energy of a reaction,
the energy barrier for
the reactants to
become products.

Rxn equilibria is linked linked to-------∆G’0

Rxn rate is linked ------------------------∆G‡

Activation energy (∆G‡): is the energy difference between that of the reactants
and a high-energy intermediate that occurs during the formation of product.
The energy that must be overcomed in order for a chemical reaction to occur
• The Gibbs free energy, G, defines the
amount of energy capable of doing work

• ΔG’0 ; standard free energy change: the free-

energy change for the reacting system
(temperature 298 K; partial pressure of each gas 1 atm, or 101.3 kPa;
concentration of each solute 1 M)
What enzymes do and do not do with
regard to biological reactions
• By lowering the activation energy, enzymes DO speed
up the rate of reactions.

• However, enzymes DO NOT supply free energy to a

reaction. Therefore, enzymes CANNOT make an
endergonic reaction proceed spontaneously. ATP
hydrolysis can be used to make an endergonic reaction
proceed spontaneously but, alone, an enzyme cannot.

• Lastly, enzymes DO NOT change the ΔG of a reaction

 Catalytic Power of Enzymes
• Enzymes are very specific, readily discriminating between
substrates with quite similar structures.
• How can these enormous and highly selective rate
enhancements be explained?
 What is the source of this energy?

1. Transient covalent interactions between enzymes and substrates

lower the activation energy (and thereby accelerate the
reaction)
Catalytic functional groups on an enzyme may form a transient
covalent bond with a substrate and activate it for reaction, or a group
may be transiently transferred from the substrate to the enzyme.

2. Noncovalent interactions between enzyme and substrate.

Much of the energy required to lower activation energies is

derived from weak, noncovalent interactions between
substrate and enzyme.
 “Lock-and-Key” model of ligand binding
• It was propounded by Emil Fischer in 1894.

• This model describes that enzyme has predetermined shape and

enzyme exhibits rigidly in its shape.

– It provides a pre-shaped and rigid template to substrate molecule.

– A substrate with complimentary conformation can fit into the enzyme

and forms enzyme-substrate complex
Limitations of Lock and Key Model

• This model could not explain competitive and

noncompetitive inhibition of enzyme.

• This model failed to explain the allosteric modulation

of enzyme activity

• An enzyme completely complementary to its

substrate would be a very poor enzyme.
 Induced fit
• In 1930, Haldane suggested that catalysis takes place in a
small region of enzyme. He called the region as “active site.”

• In 1959, Daniel E. Koshland proposed induced-fit model for

enzyme action.

• An enzyme is flexible. Its slight changes in shape (often

arising from the binding of the substrate itself) help to
position substrates for reaction after they bind

• Flexible interaction between substrate and enzyme

induces a conformational change in the protein,
which results in its increased ligand binding affinity
The “lock and key” hypothesis
can be misleading when applied
to enzymatic catalysis.

An enzyme completely
complementary to its substrate
would be a very poor enzyme.
Preferential binding of the transition state

• The enzyme is more complementary to the transition

state than do the reactants or products

• The binding of the substrate results in a distortion of

the substrate (pulling or pushing on a bond) in a way
that makes the chemical reaction easier

• Transition state stabilization: not the substrate that is

distorted but rather that the transition state
 Mechanisms of Enzyme Catalysis

Specific Catalytic groups used by enzymes to enhance

reaction rates

Once a substrate is bound to an enzyme, properly positioned

catalytic functional groups aid in the cleavage and formation of
bonds by a variety of mechanisms, including general acid-base
catalysis, covalent catalysis, and metal ion catalysis.

1. Acid/base catalysis
2. Covalent catalysis
3. Metal ion catalysis
Acid/base catalysis

Metal Ion Catalysis

Many enzymes require metal ions for maximal activity.

Metal ions:

1. Orient substrates properly for the reaction.

2. Mediate oxidation-reduction reactions within the active site.

3. Stabilize or shield negative charges.

• Enzymes that bind ATP require Mg2+ to be bound to
nucleotide (so ligand is actually Mg2+•ATP)

– shield negative charges, and

– orient the ATP substrate
Factors affecting enzyme activity
• pH of environment
• temperature
• concentration of enzyme
• concentration of substrate
• time of incubation
• coenzymes and cofactors
• inhibitors and activators
• covalent modification of enzyme
Effect of pH on Enzymatic Activity

• Enzyme-substrate recognition and catalysis are greatly

dependent on pH.

• Enzymes have a variety of ionizable side chains that

determine its secondary and tertiary structure and also affect
events in the active site.

• Enzymes are usually active only over a limited range of pH.

Enzymes have an optimum pH (or pH range) at which their
activity is maximal

• pH effects on an enzyme could cause denaturation of the

enzyme
 Effect of Temperature on Enzyme Activity
• A rise in temperature results into an increase
in rate of reaction.

• Temperature increases the kinetic energy of

substrate molecules. They can easily reach
transition state.

• At a particular temperature, rate of

enzymatic reaction is maximum and is called
as “optimum temperature.” In the human
body, optimum temperature for enzymatic
activity is 37 °C.

• A rise in 10 °C temperature doubles the rate

of enzymatic reaction until optimum
temperature

• Further increase in temperature results into

decrease in rate of reaction. It is due to
denaturation of enzyme molecules at high
temperature
Effect of Coenzymes and Cofactors

• Coenzymes are organic molecules and cofactors are metallic ions. Most of the
human body enzymes require cofactors and coenzymes for their activity.

• Coenzymes like FAD and NAD and cofactors like calcium, magnesium, and
manganese are necessary for enzyme-substrate formation.

• Rate of enzymatic activity increases in the presence of cofactors and coenzymes.

Effect of Inhibitors

• The inhibitors decrease the rate of reaction. These substances attach on active
sites, and binding of substrate molecules is decreased.

• Heavy metals like mercury, gold, and cadmium decrease rate of reaction.
Metal activated: Enzymes that bind metal ions more
weakly, only during the catalytic cycle

e.g.; Na+, K+, Mg2+, or Ca2+

Metalloenzyme :If the enzyme binds the metal very

tightly or requires the metal ion to maintain its
stable, native state

e.g. Fe2+, Fe3+, Cu2+, Zn2+,Mn2+

Enzyme Kinetics
MICHAELIS–MENTEN THEORY
To determine the rate of the reaction and how it changes in
response to changes in experimental parameters, is a discipline
known as enzyme kinetics.

1. How fast the enzyme?

2. How much affinity it has for its substrate(s)? [P]
Vo =
t
V 0 is determined for each
substrate concentration by
measuring the rate of
product formation at early
times before P accumulates

the initial rate (or initial

velocity), designated V0,
Buffer solution + same amount of when [S] is much greater
enzyme + increasing amount of substrate than the concentration of
enzyme
 Substrate Concentration Affects the Rate of Enzyme-
Catalyzed Reactions

Enzyme: constant
Substrat: increasing

Exp: enzyme: 10 mol

substrate: 0- 1.000.000—infinite mols

As a greater and greater concentration of substrate is present in the reaction

mixture, the enzyme approaches a state of saturation.
maximal velocity (Vmax): when enzyme is saturated with substrate

Vmax----changes with enzyme concentration

 Relationship of Vo to enzyme concentration:

• The rate of the reaction is directly

proportional to the enzyme concentration at
all substrate concentrations.

• For example, if the enzyme concentration is

halved, the initial rate of the reaction (vo), as
well as that of Vmax, are reduced to half that
of the original.
 The Michaelis - Menten Equation
How reaction velocity varies with substrate concentration?

•In 1913, they proposed a general

theory of enzyme action
consistent with observed enzyme
kinetics

Michaelis-Menten equation= describes the dependence of inital

velocity of an enzyme-catalyzed rxn to the substrate conc. and
to the rate constants
 Derivation of Michaelis-Menten Equation

E: enzyme k1, k-1 and k2 = rate constants

S: substrate
P: product
ES: enzyme-substrate complex
Their theory was based on the assumption:

1. Enzyme (E) and its substrate (S) associate reversibly to form

an enzyme-substrate complex, (ES)

2. Product (P) is formed in a second step when ES breaks down

to yield E+ P.

k1, k-1 and k2 = rate constants

Early in the reaction, the concentration of the product, [P], is negligible so,
reverse rxn from P to S (k-2), can be ignored.
 •The overall rate of the reaction (v) is limited by the step ES to
E+P

• This depends on 2 factors: (1) k2 and (2) the concentration of

enzyme that has substrate bound, [ES]

The rate of the enzymatic reaction (rate of formation of product) is dependent

on the concentration of the enzyme-substrate complex, [ES].

[ES] is not easily measured experimentally!

 2. Assumption of steady-state

•The concept of a steady

state was introduced by
G. E. Briggs and Haldane
in 1925.

When the enzyme is first mixed with a large excess of substrate, there is an initial
period, the pre–steady state, during which [ES] builds up. This period is usually
too short to be easily observed, lasting just microseconds. The reaction quickly
achieves a steady state in which [ES] remains aproximately constant over time.
[ES] does not change with time (the steady-
state assumption), that is, the rate of
formation of ES is equal to that of the
breakdown of ES (to E + S and to E + P).

[E (Total)] = [E(free)] + [ES]

ES is formed as rapidly from E +S

as it disappears by its two possible
fates: dissociation to regenerate E
+ S and reaction to form E + P.
•[S] >> [E]T

•over most of the reaction

course [ES] is small and does
not change

•Steady state tells us that

the rate of formation of ES is
equal to the rate of its
breakdown.

The change in concentration of ES

with time, t, is 0.
Using these assumptions, one can derive the Michaelis-Menten
Equation:
 Michaelis-Menten Equation
Equation

✓ the rate equation for a one-

substrate enzyme-catalyzed
reaction.

The rate of an enzyme-catalyzed reaction, V, at any

moment is determined by two constants, Km and Vmax,
and the concentration of substrate at that moment.
 When [S] = Km, v = Vmax/2

Vmax depends on the

amount of enzyme used in
a reaction.

✓ Km (Michaelis constant) is equal to the substrate concentration at which the

reaction rate is half its maximal value.

A numerically small (low) Km reflects a high affinity of the enzyme for substrate,
because a low concentration of substrate is needed to half-saturate the enzyme—
that is, to reach a velocity that is 1⁄2Vmax
Low Km--------high affinity
High Km--------low affinity of enzyme for substrate
• Experimentally, Km is a useful
parameter for characterizing the
number and/or types of substrates
that a particular enzyme will utilize

• It is useful for comparing similar

enzymes from different tissues or
different organisms

• In a metabolic pathway, Km values

for enzymes that catalyze the
sequential reactions may indicate
the rate limiting step for the
pathway.
Km—is characteristic of an enzyme and its particular
substrate and reflects the affinity of the enzyme for
that substrate.
Important conclusions about Michaelis-Menten
kinetics
3
1 2
 Linear Plots Can Be Derived from the Michaelis - Menten
Equation

Lineweaver—Burk (also called double reciprocal )Plot

graphically represents enzyme kinetic data.

The reciprocal (1/X) of the

Michaelis-Menten equation
 Advantages of double reciprocal plot

1. More accurate determination of Vmax , and hence

Km

2. is very useful in distinguishing between certain

types of enzymatic reaction mechanisms

3. Useful in characterizing the effects of enzyme

inhibitors
 Turnover number Kcat
• is a measure of enzymes maximal catalytic activity
– The number of reactions catalyzed by a single enzyme molecule per
second when operating at a saturating substrate concentration

Kcat =Vmax/[E] each molecule of

enzyme is
catalyzing the
formation of
... molecules of
product every
second
 Kcat and Km

• kcat and Km = both are used to evaluate the kinetic efficiency

of enzymes

–Km gives the affinity but not the velocity

–Kcat gives the velocity but not the affinity

Kcat/Km = measure of the efficiency of the catalysis

efficient enzyme = high velocities and low Km

INHIBITION OF ENZYME
ACTIVITY
Enzyme Inhibitors
• Substances that reduce or block the rate of enzymatic
reactions

• Inhibiting enzyme activity serves as a major control

mechanism in biological systems

• Many drugs and toxic agents act by inhibiting enzymes

• Inhibition by particular chemicals can be a source of insight

into the mechanism of enzyme action
 TYPES OF INHIBITION
Enzyme inhibitors
• Irreversible
–bind to enzyme covalently
• Reversible
–non-covalent binding to enzyme
• Competitive
• Non-competitive
• Uncompetitive
Enzyme-substrate complex

a competitive inhibit or binds at the active site and

thus prevents the substrate from binding

an uncompetitive inhibitor binds only to the

enzyme-substrate complex

it binds to either E or ES. Noncompetitive

inhibitor does not prevent the substrate from
binding
 Competitive Inhibition
• Inhibitor competes with the natural
substrate for the active site, occupy
the active site

• Similiar to the substrate’s structure

• Binds reversibly, thus readily

displaced

• Gives information about active site

through comparison of structures
their effect of the inhibitor is reversed by
increasing substrate concentration
 Succinate Dehydrogenase

Competitive Inhibitors
Substrates

Succinate Fumarate Malonate Glutarate Oxalate

C-OO- C-OO- C-OO-

C-OO-
H-C-H C-OO-
H-C-H C-H H-C-H
H-C-H C-OO-
H-C-H C-H C-OO-
C-OO- C-OO- H-C-H
C-OO-
 Example of a reversible inhibitors (competitive)

Dihydrofolate reductase
inhibitor
• Enzyme in nucleotide
biosynthesis (for cell division)
• folate is needed by dividing
cells

dihydrofolate
dihydrofolate reductase
X methotrexate

tetrahydrofolate
 The hallmark of competitive inhibition is that it can be overcome by a sufficiently
high concentration of substrate.

The effect of a competitive inhibitor is to increase the apparent

value of Km.
This new value of Km, called KappM ( is also shown as αKm)
 Noncompetative Inhibition
• Binds at a site distinct from the
substrate active site, but it binds
to either E or ES.

• Substrate and inhibitor do not

chemically resemble to each other

• There is no competiton in
between for binding to the
enzyme
their effect is not
reversed by adding
• a noncompetitive inhibitor does substrate
not prevent the substrate from
binding.
Noncompepetive inhibitor binds
to either E or ES. It does not
prevent the substrate from
binding
As inhibitor does not compete with the substrate for the binding site, this will
not affect the affinity of the enzyme, so Km does not change. However, Vmax
decreases. The inhibitor will decrease the amount of functional enzyme in the
reaction.
 Enzyme Inhibition (Plots)

I Competitive I Non-competitive
Vmax Vmax
vo vo
Vmax’
Direct Plots

I I

Km Km’ [S], mM Km = Km’ [S], mM

Vmax unchanged Vmax decreased
Km increased Km unchanged
Double Reciprocal

1/vo I 1/vo I

Intersect
at Y axis 1/ Vmax Intersect 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S]

Juang RH (2004) BCbasics

 Uncompetative Inhibition

Uncompetitive inhibitor binds only to

the enzyme-substrate complex
 Irreversible Inhibition
• Irreversible inhibitors bind
covalently to or destroy a
functional group on an enzyme
that is essential for the
enzyme’s activity. They also can
inhibit an enzyme by forming a
particularly stable noncovalent
association with the enzyme.

• Amino acids with key catalytic

functions in the active site can
sometimes be identified
Diisopropylfluorophosphate
(DIFP)
 Suicide inactivators (mechanism-based inactivators)

– A suicide inactivator undergoes the first few

chemical steps of the normal enzymatic
reaction, but instead of being transformed
into the normal product, the inactivator is
converted into a very reactive compound
that combines irreversibly with the enzyme

– Does not make product

– Combines irreversibly with enzyme
– also called Trojan horse substrates
– play a significant role in drug design
 Penicillin
• Penicillins block formation of the
peptide crosslinks in
peptidoglycans, acting as
mechanism-based (suicide)
inhibitors.

• covalently inhibits transpeptidase

enzyme involved in bacterial cell
wall synthesis

• transition-state analogs