Enzyme Inhibition Notes

Enzyme Inhibition

What are Enzyme Inhibitors?

Inhibitors are molecules that bind to enzymes and reduce their catalytic activity.
Two main types:
- Reversible: Can bind and release.
- Irreversible: Permanently alter enzyme activity.

Reversible Inhibitors

Three different types:
- Competitive inhibitors
- Noncompetitive inhibitors
- Uncompetitive inhibitors

Competitive Inhibitors

Bind to the same site as the enzyme substrate (active site).
Compete with the substrate for binding.
Have a chemical structure similar to the substrate.

Example:

Succinate dehydrogenase and its inhibitors: succinate (substrate), glutarate, malonate, and oxalate (competitive inhibitors).
The inhibitors have similar structures to succinate, allowing them to bind to the active site.

$C-OO^-$ $C-OO^-$ $C-OO^-$ $C-OO^-$
$|$ $|$ $|$ $|$
$C-H$ $H-C-H$ $H-C-H$ $H-C-H$
$|$ $|$ $|$ $|$
$C-H$ $H-C-H$ $H-C-H$ $C-OO^-$
$|$ $|$ $|$ $|$
$C-OO^-$ $C-OO^-$ $C-OO^-$ $C-OO^-$

Mechanism:

Enzyme (E) can bind to either the Substrate (S) or Inhibitor (I).
Inhibitor competes with substrate for the active site
E + S ⇌ ES → E + P
E + I ⇌ EI

Effect on Reaction Rate:

More substrate is needed to reach a certain reaction rate.
With enough substrate, the same $V_{max}$ is eventually reached.

Impact on Kinetics:

$V_{max}$ is unaffected.
$K_M$ appears to increase.

Lineweaver-Burk Plot:

$V_{max}$ is unaffected.
Apparent $K_M$ is increased. Intersect at Y axis.

Noncompetitive Inhibitors

Bind to a different site on the enzyme than the substrate (not the active site).
Chemical structure is different from the substrate.

Mechanism:

Inhibitor binds at a site distinct from the substrate binding site.
Does not compete for the active site.

Effect on Reaction Rate:

Once the inhibitor is bound, the enzyme is inactive.
Effectively reduces the enzyme concentration $[E]_T$ .
$V{max} = k{cat}[E]T$ ; therefore, $V{max}$ appears to decrease.

Impact on Kinetics:

Apparent $V_{max}$ is decreased.
$K_M$ is unaffected.

Lineweaver-Burk Plot:

Apparent $V_{max}$ is decreased.
$K_M$ is unaffected. Intersect at X axis.

Uncompetitive Inhibition

Inhibitor binds only to the enzyme-substrate (ES) complex, not to the free enzyme.
Both $V{max}$ and $KM$ are decreased.
Lineweaver-Burk plot shows two parallel lines.

Mechanism

$E + S \rightleftharpoons ES \longrightarrow E + P$
$\ \ \ \ \ \ \ \ i$
$\ \ \ \ \ \ \ \ \ \ S$
$\ \ \ \ \ \ \ \ \ \ \ ESI$ \
$K_{1}'$

Summary Table of Reversible Inhibitors

Property	Competitive	Noncompetitive	Uncompetitive
Effect on $V_{max}$	Unchanged	Decreased	Decreased
Effect on $K_M$	Increased	Unchanged	Decreased
Lineweaver-Burk Plot	Intersect on the y-axis	Intersect on the x-axis	Parallel lines

Clinical Insights - Enzyme Inhibitors as Drugs

Many medical drugs are enzyme inhibitors.
Methotrexate: A competitive inhibitor of dihydrofolate reductase (anti-cancer drug).
Indinavir: A competitive inhibitor of HIV protease (anti-AIDS drug).
Doxycycline: A noncompetitive inhibitor of bacterial collagenase (antibiotic for gum disease).

Irreversible Inhibitors

Permanently inactivate enzymes.
Usually form covalent bonds with the enzyme that cannot be broken.
Some used to study the active site of enzymes (e.g., Diisopropylphosphofluoridate (DIPF) reacts with serine residues).
Affinity labeling: Inhibitors resemble the substrate but contain a highly reactive chemical group, labeling residues in the active site.

Penicillin

First antibiotic drug discovered by Alexander Fleming in 1928.
Irreversibly inhibits glycopeptide transpeptidase.
The β-lactam ring reacts with a serine residue in the enzyme glycopeptide transpeptidase, permanently inactivating it.
This enzyme is essential for making the peptidoglycan of bacterial cell walls.

Kinetics of Protein-Ligand Interactions

Proteins bind to other molecules (ligands) in many situations.
Specificity of proteins for individual ligands gives proteins many of their functions, such as:
- Catalysis: enzyme-substrate binding.
- Recognition: cell surface receptor-signal molecule binding.
- Transport: membrane transport protein-metabolite binding.

Hyperbolic Kinetics (Michaelis-Menten)

Binding of protein to its ligand shows hyperbolic kinetics.
Graph of activity against ligand concentration is hyperbolic in shape.
Becomes saturated.
Greatest increase in activity for a small increase in ligand concentration occurs at very low ligand concentrations.
Many enzymes show hyperbolic kinetics.

Sigmoidal Kinetics

Binding of protein to its ligand shows sigmoidal kinetics.
Graph of activity against ligand concentration is sigmoidal in shape (roughly S-shaped).
Becomes saturated.
Greatest increase in activity for a small increase in ligand concentration occurs at intermediate/low ligand concentrations.
Many enzymes show sigmoidal kinetics – particularly those controlling the committed step in a pathway.

Characteristics of Proteins Showing Sigmoidal Kinetics

Composed of two or more subunits.
The subunits can exist in two different shapes.
When the target ligand binds to the binding site on one of the subunits, it increases the affinity of the other subunit(s) for the ligand.
This is described as cooperative binding or cooperativity.
It gives rise to the sigmoidal curve.
Allosteric enzymes are only one of the groups of proteins that show sigmoidal kinetics.

Cooperativity

At a low ligand:protein ratio, most proteins have only one or no ligand bound (ligand binding at low affinity).
As the ligand:protein ratio increases, proteins are increasingly binding a second ligand with greater affinity than the first.
Eventually, the protein becomes saturated with the ligand.