Enzyme Kinetics - Inhibition

Enzyme Inhibitors
• Enzyme inhibitors are the compound which may
bind to the enzyme and reduce their activity.
• Enzyme Inhibitors:
– Reversible (easily unbinds)
– Irreversible (stable complex)
• Irreversible inhibitors – reversed – chelating
agents (EDTA or citrate)
 Reversible Inhibitors
Three major classes:
– Competitive Inhibition
– Noncompetitive Inhibition
– Uncompetitive Inhibition
– Substrate Inhibition

General Michaelis-Menten Equation

This form of the Michaelis-Menten equation can

be used to understand how each type of inhibitor
affects the reaction rate curve.
Competitive Inhibition

In competitive inhibition, the

inhibitor (substrate analogs)
competes with the substrate
for the same binding site

Competitive Inhibition
- Reaction Mechanism

In competitive inhibition, the

inhibitor binds only to the
free enzyme, not to the ES
complex
 M-M Kinetics
Assuming rapid equilibrium:

K’m, app

Competitive inhibitors alter the

apparent Km, not the Vmax

Vmax,app = Vmax
Km,app > Km
 The Lineweaver-Burk plot is
diagnostic for competitive inhibition

Noncompetitive Inhibition
• NCI are not
substrate analogs.
• The inhibitor does
not interfere with
substrate binding
(and vice versa)
• Binds on sites other
than the active
sites.
 Noncompetitive Inhibition -
Reaction Mechanism

In noncompetitive
inhibition, the
inhibitor binds
enzyme regardless
of whether the
substrate is bound

Noncompetitive Inhibition
Defining:

The rate equation will be:

Or
 Noncompetitive inhibitors decrease
the Vmax,app, but don’t affect the Km

Vmax,app < Vmax

Km,app = Km

The Lineweaver-Burk plot is diagnostic

for noncompetitive inhibition
Uncompetitive Inhibition

In uncompetitive
inhibition, the
inhibitor binds
only to the ES
complex

Uncompetitive Inhibition -
Reaction Mechanism

In uncompetitive
inhibition, the
inhibitor binds only
to the ES complex,
it does not bind to
the free enzyme
 Uncompetitive Inhibition

Uncompetitive inhibitors decrease

both the Vmax,app and the Km,app
Vmax,app < Vmax
Km,app < Km
Notice that at low substrate
concentrations,
uncompetitive inhibitors
have little effect on the
reaction rate because the
lower Km, app of the enzyme
offsets the decreased Vmax,app
 The Lineweaver-Burk plot is
diagnostic for uncompetitive inhibition

Irreversible Inhibition
In irreversible
inhibition, the
inhibitor binds to the
enzyme irreversibly
through formation of
a covalent bond with
the enzyme ,
permanently
inactivating the
enzyme
 Irreversible Inhibition - Reaction
Mechanism

In irreversible inhibition,
the inhibitor permanently
inactivates the enzyme.
The net effect is to remove
enzyme from the reaction.
Vmax decreases
No effect on Km
E~I

The Michaelis-Menten plot for an irreversible

inhibitor looks like noncompetitive inhibition

Vmax,app < Vmax

Km,app = Km
 Irreversible inhibition is distinguished from
noncompetitive inhibition by plotting Vmax vs [E]t

Enzyme is
inactivated
until all of the
irreversible
inhibitor is
used up

Examples of Irreversible Inhibitors

• Cholinesterase (ko-li-nes-ter-ace) is one of many
important enzymes needed for the proper functioning
of the nervous systems of humans, other vertebrates,
and insects.
• Certain chemical classes of pesticides, such as
organophosphates (OPs) and carbamates (CMs) work
against undesirable bugs by interfering with, or
'inhibiting' cholinesterase.
• While the effects of cholinesterase inhibiting products
are intended for insect pests, these chemicals can also
be poisonous, or toxic, to humans in some situations.
 Penicillin is a suicide inhibitor

• Glycopeptide transpeptidase catalyzes the formation of cross-links in the cell

walls of bacteria.
• This enzyme also catalyzes the reverse reaction, the hydrolysis of peptide
bonds.
• During the course of hydrolyzing the strained peptide bond in penicillin, the
enzyme activates the inhibitor (penicillin), which then covalently modifies an
active site serine in the enzyme.
• In effect, the enzyme “commits suicide” by hydrolyzing the strained peptide
bond in penicillin.

Summary-Enzyme Inhibition
• Competitive Inhibitor
– Binds to substrate binding site
– Competes with substrate
– The affinity of the substrate appears to be decreased
when inhibitor is present (Km,app >Km)
• Noncompetitive inhibitor
– Binds to allosteric site
(allosteric enzyme-more than one substrate binding site,
property – allostery/cooperative binding)
– Does not compete with the substrate for binding to
the enzyme
– The maximum velocity appears to be decreased in
the presence of the inhibitor (Vmax,app <Vmax)
• Uncompetitive Inhibitor
– Binds to the enzyme only after the substrate has
bound
– The affinity of the substrate appears to be increased
and the maximum velocity appears to be decreased
when inhibitor is present (Km,app <Km,
Vmax,app <Vmax),
• Irreversible Inhibitor
– Covalently modifies and permanently inactivates the
enzyme