Lecture 6 - Enzymes As Drug Targets

Enzymes as Drug Targets
Drugs that Target Enzymes

Structure and function of enzymes
Lowering the activation energy of reaction
Energy Energy
Transition state New
transition
Act. state
energy Act.
energy
Starting Starting
∆G ∆G
material material
Product Product
WITHOUT ENZYME WITH ENZYME
• Enzymes lower the activation energy of a reaction but DG

remains the same
Structure and function of enzymes
Methods of enzyme catalysis
• Provide a reaction surface (the active site)

• Provide a suitable environment (hydrophobic)
• Bring reactants together
• Position reactants correctly for reaction
• Weaken bonds in the reactants
• Provide acid / base catalysis
• Provide nucleophiles
• Entropy
Catalysis mechanisms
Acid/base catalysis
• Histidine +H
NH NH
N -H N
H
Non-ionised Ionised
Acts as a basic catalyst Acts as an acid catalyst
(proton 'sink') (proton source)
Nucleophilic residues
H H
H3N CO2 H3N CO2
L-Serine L-Cysteine
OH SH
Catalysis mechanisms
Serine acting as a nucleophile
Substrate
H 2O
HO Product
OH O OH
Ser Ser Ser

The Effect of Substrate Concentration
on Reaction Velocity
The Michaelis-Menten Equation
Simplification of kinetic scheme (by rapid equilibrium or steady state approaches) leads to the
Michaelis-Menten equation.
vo = k2[E]t[S]/(KM + [S])
or
vo = Vmax[S]/(KM + [S])
(since Vmax = k2[E]t when [S] >> KM)

Km
• Km is, under true Michaelis-Menten
conditions, an estimate of the dissociation
constant of E from S
• Small Km means tight binding; high Km
means weak binding
• Below KM, enzymes are sensitive to
substrate concentration
• Above KM, substrate concentrations have
little effect on enzyme activity.
Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmax is the theoretical maximal rate of the reaction -
but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is asymptotically approached as substrate is
increased
Lineweaver-Burk Plot
1 𝐾𝑚 1 1
= × +
𝑉0 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥
A plot of 1/Vo
12
Enzyme Inhibition - Four mechanistic categories
1. Competitive inhibition. Inhibitor competes reversibly with substrate for the

active site.
2. Uncompetitive inhibition. Inhibitor binds only to the ES complex, leading

to EIS intermediates. This is very rare.
3. Non-competitive (reversible) inhibition. Inhibitor binds non-

covalently to sites other than the active site (Allosteric inhibition).
4. Irreversible inhibition. Inhibitor binds covalently.

1. Competitive Inhibitors
• Competitive inhibitors compete with substrate for the active site

of the enzyme.
• They are structurally similar to normal substrate for the enzyme
and bind reversibly to the active site.
• The enzyme can bind either substrate or the competitive
inhibitor but not both
• High substrate concentrations reverse inhibition
14
Lineweaver-Burk Plot of Competitive Inhibition
KM is increased
Vmax is unchanged
Vmax
-1/km
15
Example of competitive inhibitor
• Ethylene glycol and methanol are toxic

• Ethylene glycol and methanol are substrates for alcohol dehydrogenase
• Ethylene glycol is eventually converted to oxalic acid (toxic)
• Methanol is eventually converted to formic acid (toxic)
• A new treatment, approved by the FDA in December 1997, uses the alcohol
dehydrogenase inhibitor 4-methylpyrazole (trade name = Antizol).
• Unlike ethanol, 4-methylpyrazole is not a substrate for the enzyme, and is
therefore not metabolized
4-methylpyrazole – used as an antidote for ethylene glycol and methanol poisoning

2. Uncompetitive Inhibitors
• Substrate binds to the enzyme’s active site to

form ES complex.
• Uncompetitive inhibitor binds to the ES

complex, blocks formation of product.
• An uncompetitive inhibitor only binds to ES complex resulting in lower KM

and better enzyme-substrate binding
• Works best at high substrate concentration
• Very rare
17
Lineweaver-Burke Plot of Uncompetitive Inhibition
KM is decreased
Vmax is also decreased
18
Example of uncompetitive inhibitor
• 5-a-reductase converts testosterone to DHT

• DHT leads to benign prostatic hyperplasia (BPH, enlargement of prostate);
promotes prostate growth
• Finasteride inhibits 5-a-reductase
Finasteride
3. Noncompetitive Inhibitors
1. A molecule binds to the enzyme on an
allosteric site and not the active site.
2. This causes a change in the enzyme's 3D
structure.
3. The active site still binds substrate (no
change in affinity).
4. The ES complex is no longer in the optimal
arrangement to stabilize the transition state
and Vmax is lowered.
The effects of a noncompetitive inhibitor cannot be reversed by increasing the

substrate concentration
20
Lineweaver-Burk Plot of Noncompetitive Inhibition
KM is unchanged
Vmax is decreased
21
Example of non-competitive inhibitor
• Reverse transcriptase is essential for replication of HIV

• Nevirapine is a non-nucleoside reverse transcriptase inhibitor (NNRTI)
Nevirapine
Ki, the inhibitor constant
The inhibitor constant, Ki, is an indication of how
potent an inhibitor is; it is the concentration
required to produce half maximum inhibition.
Plotting 1/v against concentration of inhibitor at
each concentration of substrate (the Dixon plot)
gives a family of intersecting lines.
For a competitive inhibitor, the lines converge

above the x axis, and the value of [I] where they
intersect is -Ki
Ki, the inhibitor constant
For a non-competitive inhibitor, the lines

converge on x axis, and the value of [I] where
they intersect is -Ki
Cheng-Prusoff equation
IC50 curve
4. Irreversible inhibitors
X
Covalent Bond
OH OH O
Irreversible inhibition
• Inhibitor binds irreversibly to the active site

• Covalent bond formed between the drug and the enzyme
• Substrate is blocked from the active site
• Increasing substrate concentration does not reverse inhibition
• Inhibitor likely to be similar in structure to the substrate
Electrophilic functional groups
Example of irreversible inhibitor
Suicide substrates
• aka Mechanism-based inhibition

• Type of irreversible inhibition
• Inhibitor is “activated” by enzyme
at the active site (advantage)
• E.g. Clavalunic acid/b-lactamase
Transition state analogues
Angiotensinin II – increase in bp

Lecture 6 - Enzymes As Drug Targets

Uploaded by

Copyright:

Available Formats

Lecture 6 - Enzymes As Drug Targets

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Lecture 6 - Enzymes As Drug Targets

Uploaded by

Copyright:

Available Formats

Enzymes as Drug Targets

Drugs that Target Enzymes

WITHOUT ENZYME WITH ENZYME

• Enzymes lower the activation energy of a reaction but DG

• Provide a reaction surface (the active site)

Serine acting as a nucleophile

Ser Ser Ser

(since Vmax = k2[E]t when [S] >> KM)

1. Competitive inhibition. Inhibitor competes reversibly with substrate for the

2. Uncompetitive inhibition. Inhibitor binds only to the ES complex, leading

3. Non-competitive (reversible) inhibition. Inhibitor binds non-

4. Irreversible inhibition. Inhibitor binds covalently.

• Competitive inhibitors compete with substrate for the active site

• Ethylene glycol and methanol are toxic

4-methylpyrazole – used as an antidote for ethylene glycol and methanol poisoning

• Substrate binds to the enzyme’s active site to

• Uncompetitive inhibitor binds to the ES

• An uncompetitive inhibitor only binds to ES complex resulting in lower KM

• Works best at high substrate concentration

• 5-a-reductase converts testosterone to DHT

The effects of a noncompetitive inhibitor cannot be reversed by increasing the

• Reverse transcriptase is essential for replication of HIV

For a competitive inhibitor, the lines converge

For a non-competitive inhibitor, the lines

• Inhibitor binds irreversibly to the active site

• aka Mechanism-based inhibition

You might also like