6.

1 Enzymes are effective Biological Catalysts:

• Probably the most important function of proteins

is catalysis.
• Biological catalysts are called enzymes.
• Enzymes are globular Proteins.
• With the exception of some recently discovered
RNAs (Ribozymes) that have catalytic activity, all
enzymes are globular proteins.
• Enzymes are the most efficient catalysts known.
6.2 Kinetics Versus Thermodynamics:
• Catalysts speed up a reaction by lowering the activation
energy, a kinetic parameter.
• They do not affect the thermodynamics of the reaction (∆ Gº).
• They help the substrate and Enzyme to attain the transition
state; the high point on an energy diagram for the reaction.
Activation energy profiles:

(a)The activation energy profile

for a typical reaction. The
reaction shown here is
exergonic (energy-releasing).

(b) A comparison of activation

energy profiles for catalyzed
and uncatalyzed reactions.
The activation energy of the
catalyzed reaction is much
less than that of the
uncatalyzed reaction.
6.3 Enzyme Kinetic Equation:

• We can describe the kinetic aspects of

reactions by rate equations.
• In such equations, we define reaction rate in
terms of the disappearance of reactants or the
appearance of products.
• We can describe rates in terms of changes in
the concentrations of reactants or products as
a function of time.
Rate = k[A]f[B]g
K: Rate Constant
f & g: Exponents determined experimentally
• First Order reaction: A P
Rate = k[A]1

• Second Order reaction: A + B C+D

Rate = k[A]1[B]1

• Zero Order reaction: very rare, occurred when the

substrate concentration is so high and the enzyme
is completely saturated with reactant molecules.
A B
Rate = k[A]0 = k
 6.4 Enzyme Substrate Binding
• The first step in an enzyme-catalyzed reaction is the binding of
the enzyme to the substrate to form a complex (ES complex)
• The formation of the complex leads to formation of the
transition-state species, which, in turn, forms the product.
• A substrate binds to a small portion of the enzyme called the
active site.

• Two models have been proposed to describe enzyme–substrate

binding:
a- the lock-and-key model, in which there is an exact fit
between the enzyme and substrate.
b- the induced-fit model, in which the enzyme is considered
to have conformational flexibility and there is an exact fit only
when the substrate is bound.
• The active site of an enzyme forces the substrate to mimic the
transition state of the reaction, which is the primary way the
activation energy of the reaction is lowered.
Formation of product from substrate (bound to the enzyme ES),
followed by release of the product.

A B
6.5 Example of Enzyme-Catalyzed Reaction
• In some enzyme-catalyzed reactions, the rate of
reaction rises as substrate concentration
increases and then levels off.
• When this behavior is shown on a graph, the
curve is hyperbolic.
• e.g. Chymotrypsin is a digestive enzyme that
shows this behavior.
• In other reactions, the shape of the curve is
Sigmoidal: S-shape characterized by cooperative
interactions.
• e.g Aspartate transcarbamoylase is an enzyme
involved in the production of pyrimidines, and it
shows sigmoidal kinetics.
Hyperbolic Sigmoidal
 Factors Affecting Reaction Velocity

Substrate concentration
1.Maximal velocity:
•Rate of enzyme catalyzed reaction increases with substrate
concentration until a maximal velocity (Vmax) is reached
•Leveling off of the reaction rate at high substrate concentrations
reflects the saturation with substrate of all available binding sites on
the enzyme molecules present
. Temperature:
1.Increase of velocity with temperature
•Reaction velocity increases with temperature until a peak velocity is
reached
•Increased number of molecules having sufficient energy to pass over the
energy barrier
2. Decrease of velocity with higher temperature
•Further elevation of temperature results in a decrease in reaction
velocity as a result of temperature-induced denaturation of the enzyme
C. pH
1.Effect of pH on the ionization of the active site:
•Conc. of H+ ions affects reaction velocity in several ways
•Catalytic process usually requires that the enzyme and substrate have
specific chemical groups in either an ionized or unionized state in
order to interact
The effect of temperature
on enzyme activity:
• The relative activity of
an enzymatic reaction as a
function of temperature.
• The decrease in activity
above 50°C is due to
thermal denaturation.
2. Effect of pH on enzyme denaturation:

•Extremes of pH can also lead to denaturation of the enzyme, because the

structure of the catalytically active protein molecule depends on the ionic
characteristics of the amino acid side chains
3. The pH optimum varies for different enzymes:
•The pH at which maximal enzyme activity is achieved is different for different
enzymes, and often reflects the (H+) at which the enzyme functions in the body
Michaelis- Menten Equation
A.Reaction model:
•The enzyme reversibly combines with its substrate to form an ES
complex that subsequently breaks down to product, regenerating the
free enzyme
B. Michaelis-Menten Equation
Describes how reaction velocity varies with substrate concentration

•Relative concentration of E and S: The concentration of substrate (S)

is much greater than the concentration of enzyme (E), so the total
substrate bound by the enzyme at any one time is small
•Steady-State assumption: Intermediate in a series of reactions is said
to be in steady-state when its rate of synthesis is equal to its rate of
degradation
•Initial velocity: Only initial reaction velocities (vo) are used in the
analysis of enzyme reactions. This means that the rate of reaction is
measured as soon as enzyme and substrate are mixed
Important conclusions about Michaelis-Menten kinetics

Characteristics of Km: Km-the Michaelis Constant – is characteristic

of an enzyme and its particular substrate, and reflects the affinity of the
enzyme for that substrate
•Km is numerically equal to substrate concentration at which the
reaction velocity is equal to ½ Vmax
•Km does not vary with the concentration of enzyme
1.Small Km: High affinity of the enzyme for substrate
2.Large Km: Low affinity of the enzyme for substrate
2. Relationship of velocity to enzyme concentration:

•Rate of reaction is directly proportional to enzyme

concentration at all substrate concentrations
3. Order of Reaction:
•When (S) is much less than Km the velocity of the reaction
is approximately proportional to the substrate concentration
(First Order)
•When (S) is much greater than Km the velocity is constant
and equal to Vmax . The rate of reaction is then independent
of substrate concentration (Zero Order)
6.6 The Michaelis-Menten Approach to Enzyme Kinetics

• Michaelis and Menten developed a series of

mathematical relationship to explain the behavior of
many nonallosteric enzymes.
• Michaelis-Menten equation describes several
parameters, including the maximum velocity Vmax and
the Michaelis Constant KM.
• Vmax: Velocity at enzyme saturation level.
• KM = [substrate] that generate half Vmax.
• Steady-State Theory:
ES formation = ES breakdown.
The rate and the observed kinetics of an enzymatic reaction depend on substrate
concentration. The concentration of enzyme, [E], is constant.
 What does Km mean?
1. Km = [S] at ½ Vmax
2. Km is a combination of rate constants describing
the formation and breakdown of the ES complex
3. Km is usually a little higher than the
physiological [S]
 What does Km mean?
4. Km represents the amount of substrate required to
bind ½ of the available enzyme (binding constant of
the enzyme for substrate)
5. Km can be used to evaluate the specificity of an
enzyme for a substrate (if it obeys M-M)
6. Small Km means tight binding; high Km means weak
binding

Glucose Km = 8 X 10-6
Hexose Kinase Allose Km = 8 X 10-3
Glucose + ATP <-> Glucose-6-P + ADP Mannose Km = 5 X 10-6
 V max
0.25
B
B B [S] Vo
0.2 B 0.5 0.075
0.75 0.09
2 0.152
0.15 B
4 0.196
Vo

6 0.21
0.1 8 0.214
B 10 0.23
B
0.05
Km
Km ~ 1.3 mM
0
0 1 2 3 4 5 6 7 8 9 10 Vmax ~ 0.25
[S]
 Turnover number
• Vmax= kcat [ E]total
• Kcat =Vmax / [E]total
• At high conc. Of substrate = the velocity of the rxn is Vmax
• SO The rate of rxn is determined by E concentration, and the rate constant is
called CATALYTIC CONSTANT

• K cat = maximum No. of molecules converted to product each

second per mole of enzyme or the maximum No. of substrate
converted to product each second by each active site of the E.

THIS IS CALLED TURNOVER NUMBER

Important conclusions about Michaelis-Menten kinetics
Lineweaver-Burke Plot
Inhibition of Enzyme Activity
•When Vo is plotted against [S], it is not always possible to
determine when Vmax has been achieved, because of the
gradual upward slope of the hyperbolic curve at high
substrate concentrations

•However, if 1/Vo is plotted versus 1/[S], a straight line is

obtained
•This plot, the Lineweaver-Burke plot (also called a double-
reciprocal plot) can be used to calculate Km and Vmax, as
well as to determine the mechanism of action of enzyme
inhibitors.
Vmax = velocity where all of the enzyme
is bound to substrate
(enzyme is saturated with S)

Km = [S] @ ½ Vmax
(units moles/L=M)
(1/2 of enzyme bound to S)
• Michaelis- Menton Equation
• Describes rectangular hyperbolic plot
• Vo = Vmax [S]
Km + [S]
Lineweaver-Burk Plots
(double reciprocal plots)

•Plot 1/[S] vs 1/Vo

•L-B equation for straight line
•X-intercept = -1/Km
•Y-intercept = 1/Vmax
•Easier to extrapolate values w/
straight line vs hyperbolic curve
A Lineweaver–Burk double reciprocal plot of enzyme kinetics.
The reciprocal of reaction velocity, 1/V, is plotted against the reciprocal of the substrate
concentration, 1/[S]. The slope of the line is KM/Vmax, and the y intercept is 1/Vmax.
The x intercept is –1/KM.
•Inhibitor: Any substance that can diminish
the velocity of an enzyme catalyzed reaction
•Reversible Inhibitors: bind to enzyme
through noncovalent bonds. Dilution of the
enzyme inhibitor complex results in
dissociation of the reversibly bound inhibitor
and recovery of enzyme activity
•Irreversible Inhibitors: occurs when
inhibited enzyme does not gain activity on
dilution of the enzyme inhibitor complex
 Enzyme Inhibition
• Inhibitor – substance that binds to an enzyme and
interferes with its activity
• Can prevent formation of ES complex or prevent ES
breakdown to E + P.
Irreversible and Reversible Inhibitors
• Irreversible inhibitor binds to enzyme through covalent
bonds (binds irreversibly)
• Reversible Inhibitors bind through non-covalent
interactions (disassociates from enzyme)
• Why important?
Competitive Inhibitors look like substrate
O
O

HO C NH2 H2N S NH2

PABA Sulfanilamide
PABA precursor to folic acid in bacteria
Para-Aminobenzoic Acid

O2C-CH2-CH2-CO2 -------> O2C-CH=CH-CO2

succinate fumarate

Succinate dehydrogenase

O2C-CH2-CO2
Malonate
 Competitive Inhibitor (CI)

CI binds free enzyme

Competes with substrate for enzyme binding.
•Raises Km without affecting Vmax
•Can relieve inhibition with more S
A Lineweaver–Burk double-reciprocal plot of enzyme kinetics
for competitive inhibition
Competitive inhibition
Inhibitor binds reversibly to the same site that the substrate would
normally occupy and therefore competes with the substrate for that site
E.g. Atorvastatin and simvastatin
 Non-competitive Inhibitor (NI)

•NI can bind free E or ES complex

•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t affect Km
•Alters conformation of enzyme to affect catalysis but not substrate
binding
A Lineweaver–Burk plot of enzyme kinetics for noncompetitive
inhibition.
 Noncompetitive Inhibition

Inhibitor and substrate bind at different sites on the enzyme

E.g.Binding of lead with Ferrochelatase
Modes of action of inhibitors.
(A) and (B): The distinction between competitive
and noncompetitive inhibitors is that a competitive
inhibitor prevents binding of the substrate to the
enzyme, whereas a noncompetitive inhibitor does
not.
(c) A noncompetitive inhibitor binds at a site other
than the active site.
The substrate still binds, but the enzyme cannot
catalyze the reaction because of the presence of the
bound inhibitor
 Uncompetitive Inhibitor (UI)

•UI binds ES complex

•Prevents ES from proceeding to E + P or back to E + S.
•Lowers Km & Vmax, but ratio of Km/Vmax remains the same
•Occurs with multisubstrate enzymes
AN UNCOMPETITIVE INHIBITOR BINDS AT A
SITE DISTINCT FROM THAT OF THE
SUBSTRATE ACTIVE SITE AND UNLIKE A
NONCOMPETITIVE INHIBITOR BINDS ONLY
TO ES COMPLEX
Mixed inhibition is a type of enzyme inhibition in which the
inhibitor may bind to the enzyme whether or not the enzyme has
already bound the substrate but has a greater affinity for one state
or the other. It is called "mixed" because it can be seen as a
conceptual "mixture" of competitive inhibition, in which the
inhibitor can only bind the enzyme if the substrate has not already
bound, and uncompetitive inhibition, in which the inhibitor can
only bind the enzyme if the substrate has already bound. If the
ability of the inhibitor to bind the enzyme is exactly the
same whether or not the enzyme has already bound the substrate,
it is known as a non-competitiveinhibitor. Non-competitive
inhibition is sometimes thought of as a special case of mixed
inhibition.
In mixed inhibition, the inhibitor binds to an allosteric site, i.e. a
site different from the active site where the ubstrate binds.
However, not all inhibitors that bind at allosteric sites are mixed
inhibitors.
 Mixed Inhibition
• It is called "mixed" because it can be seen as a
conceptual "mixture" of competitive
inhibition, in which the inhibitor can only bind
the enzyme if the substrate has not already
bound,
• and uncompetitive inhibition, in which the
inhibitor can only bind the enzyme if the
substrate has already bound. If
 Irreversible Inhibitors

CH3 F CH3 O

H C O P O C H S C O CH2CH3
CH3 O CH3
malathion
H3C O P S C H

Diisopropyl fluorophosphate O CH2

(nerve gas) CH3 C O CH2CH3

H3C O P S NO2

CH3 Organophosphates
•Inhibit serine Hydrolases, and one of
parathion
them is:
•Acetylcholinesterase
 Active site of VX-478 complexed
with HIV-1 protease.

Structure of amprenavir
(VX-478), an HIV protease
inhibitor developed by
Vertex Pharmaceuticals
Regulation of Enzyme Activity