13- Enzyme Inhibition

What two aspects of enzyme activity can an inhibitor alter?

Substrate binding and the enzyme's ability to convert substrate into product (turnover number)

How could an inhibitor interfere with catalysis without preventing substrate binding?

It could allow the substrate to bind but reduce the enzyme–substrate complex's ability to form product.

Irreversible inhibitor

A substance that causes inhibition that cannot be reversed

• Usually involves formation or breaking of covalent bonds to or on the enzyme (modifies the enzyme)

What is another name for irreversible inhibitors?

Inactivators

Reversible inhibitor

A reversible inhibitor binds to an enzyme to inhibit it but can later dissociate, allowing the enzyme to regain activity

• Usually interact with enzymes through noncovalent interactions, such as hydrogen bonds, ionic attractions, and hydrophobic interactions

What are the 3 types of inhibition?

1. Competitive

2. Uncompetitive

3. Mixed or Noncompetitive

What two places may an inhibitor bind?

1. At the same binding site as one of the substrates

• The inhibitor must structurally resemble the substrate

2. At an alternative site, affecting catalytic activity without affecting substrate binding

What is competitive inhibition?

A competitive inhibitor competes with the substrate for the same binding site on the enzyme

• E + I ⇌ EI

What is structural mimicry?

When an inhibitor resembles a substrate closely enough to bind at the enzyme's substrate-binding site

How do competitive inhibitors affect a reaction?

Enzymes are temporarily occupied by inhibitors, so there are fewer free enzymes available to form an ES complex

What does K_I represent?

The inhibitor dissociation constant, which describes the binding of inhibitor to enzyme.

Formula for K_I:

What does a small K_I indicate?

The inhibitor binds tightly to the enzyme

What does a large K_I indicate?

There is weak binding between the inhibitor and the enzyme

• It is easy for EI to dissociate

Why does competitive inhibition increase apparent K_M?

The inhibitor occupies free enzyme, so a higher substrate concentration is needed to achieve a given velocity.

What is apparent K_M?

Apparent K_M is the K_M value you observe experimentally when something else, such as an inhibitor or another molecule, affects the enzyme's behavior.

Abbreviations for apparent K_M:

K_{M, app} or αK_M

• K_{M, app} = αK_M

Michaelis-Menten equation with apparent K_M:

What does α mean in terms of apparent K_M?

Alpha is a factor that tells you how much a competitive inhibitor increases the apparent K_M

• It depends on the inhibitor concentration and how strongly the inhibitor binds to the enzym

Formula for α (apparent K_M)

How do you overcome the effects of a competitive inhibitor?

Increase [S]

• This allows the substrate to outcompete the inhibitor for the binding sit

What is trypsin's biological function?

It is a digestive enzyme that cleaves peptide bonds in proteins

Why can benzamidine act as a competitive inhibitor of trypsin?

Benzamidine resembles the arginine side chains of Trypsin, so it can occupy the trypsin's substrate-binding pocket

Why does benzamidine not behave like a normal substrate when occupying the trypsin's substrate-binding pocket?

It lacks the normal peptide-bond arrangement needed for the usual cleavage reaction

• Looks like a substrate but doesn’t act like a substrate!

Why does competitive inhibition leave k_cat unchanged in the basic model?

The inhibitor affects substrate access, not the catalytic turnover of enzyme molecules that have bound substrate

• For a reversible competitive inhibitor, the enzyme returns to its normal catalytic activity once the inhibitor detaches.

How does competitive inhibition affect apparent k_cat/ K_M?

t decreases the apparent value because apparent K_M increases while k_cat stays the same.

What happens to the slope of a Lineweaver–Burk plot during competitive inhibition?

It increases

• because the apparent K_M increases, while V_max stays the same

• REMEMBER: Slope= K_M/ V_max

What happens to the y-intercept during competitive inhibition?

It stays the same because V_max is unchanged.

• REMEMBER: y-intercept= V_max

Why is competitive inhibition called an intersecting pattern?

The lines with and without inhibitor meet at the same y-intercept.

What happens to the x-intercept during competitive inhibition?

It moves closer to 0 (more and more negative as α increases)

• REMEMBER: x-intercept = -1/ αK_M for competitive inhibitors and -1/ K_M for no inhibition

Formula for finding K_I of competitive binding by comparing K_M with and without an inhibitor:

Formula for finding K_I of competitive binding by comparing Lineweaver–Burk slopes with and without an inhibitor:

What is uncompetitive inhibition?

An uncompetitive inhibitor binds to the enzyme–substrate complex, ES, but does not bind to the free enzyme in the basic model.

• ES + I ⇌ ESI

What happens to Vmax during uncompetitive inhibition?

It decreases

• Why? The inhibitor binds to the enzyme–substrate complex (ES), preventing it from producing product.

What happens to K_M during uncompetitive inhibition?

It decreases

• Why? The inhibitor binds to ES and traps it as ESI, stabilizing the substrate-bound state and decreasing the apparent K_M, even though product formation is inhibited.

Why can high substrate concentrations not overcome uncompetitive inhibition?

The inhibitor binds the ES complex, so increasing substrate does not prevent inhibitor binding to that complex

Why does V_max/ K_M remain unchanged in pure uncompetitive inhibition?

The inhibitor does not affect ES complex formation

Michaelis-Menten equation for uncompetitive inhibition:

What is α’ regarding uncompetitive inhibition?

Alpha prime (α’) tells you how much an uncompetitive inhibitor affects the enzyme's kinetics by binding to the enzyme–substrate complex (ES).

α’ formula:

K_{M, app} formula for uncompetitive inhibition:

V_{max, app} for uncompetitive inhibition:

Why does uncompetitive inhibition reduce apparent k_cat?

Some enzyme–substrate complexes are trapped as inactive ESI rather than producing product

What happens to k_cat/K_M in pure uncompetitive inhibition?

It remains unchanged in the basic model because ES must be formed prior to the inhibitor binding

Why can uncompetitive inhibition become more pronounced as substrate concentration increases?

More ES complexes form, creating more targets for the inhibitor.

Formula for finding K_I of uncompetitive inhibition by comparing y-intercepts”

Formula for finding K_I of uncompetitive inhibition by comparing x-intercepts”

What is Mixed or Noncompetitive Inhibition

An inhibitor can bind to both the free enzyme, E, and the enzyme–substrate complex, ES

What is mixed inhibition?

An inhibitor binds both free enzyme (E) and enzyme-substrate complex (ES) with different affinities

What is pure noncompetitive inhibition?

An inhibitor binds both free enzyme (E) and enzyme-substrate complex (ES) with the same affinities

What happens to V_max during mixed inhibition?

Decreases

• The inhibitor reduces the amount of enzymes available to produce product

What happens to K_M during mixed inhibition?

Can increase or decrease depending on whether the inhibitor prefers E or ES

• Prefers ES —> K_M decreases

• Prefers E —> K_M increases

What happens to V_max during pure noncompetitive inhibition?

It decreases because the inhibitor reduces the enzyme's ability to produce product.

What happens to K_M during pure noncompetitive inhibition?

It stays the same because the inhibitor binds E and ES equally well.

Michaelis- Menten equation for mixed or non-competitive inhibition:

α formula for mixed or non-competitive inhibition:

K_I: inhibitor binding to free enzyme

K_I’: inhibitor binding to the enzyme–substrate complex

K_{M, app} formula for mixed or non-competitive inhibition:

V_max formula for mixed or non-competitive inhibition:

Finding K_I of mixed or noncompetitive inhibition by comparing slopes:

Finding K_I of mixed or noncompetitive inhibition by comparing y-intercepts:

What is the general Lineweaver–Burk pattern for mixed inhibition?

The lines intersect to the left of the y-axis

Why is pure noncompetitive inhibition considered a special case of mixed inhibition?

It occurs when the inhibitor's affinity for free enzyme and ES is exactly equal

What happens to V_max when an irreversible inhibitor inactivates some enzyme molecules?

It decreases because fewer functional enzyme molecules remain

What happens to K_M when an irreversible inhibitor inactivates some enzyme molecules?

The surviving active enzymes retain their original substrate-binding properties

Why can irreversible inhibition resemble pure noncompetitive inhibition on a Lineweaver–Burk plot?

Both can decrease V_max while leaving K_M unchanged

How do irreversible inhibitors modify an enzyme?

Many irreversible inhibitors form a covalent bond with the enzyme

• This changes the enzyme chemically and prevents it from functioning normally.

Why can’t you distinguish Lineweaver-Burk plots of irreversible inhibitors from the noncompetitive inhibition?

Lineweaver-Burk plots of irreversible inhibitors cannot be distinguished from the noncompetitive inhibition pattern because of the loss of active enzyme

What happens to enzyme activity after an irreversible inhibitor is removed from the enzyme’s binding site?

With an irreversible inhibitor, removing free inhibitor generally does not restore activity because the enzyme has already been chemically modified or inactivated

• This is time-dependent (how long was the inhibitor bound?)

How does TPCK inactivate chymotrypsin?

It forms a covalent bond with His 57, preventing the enzyme from functioning normally

Why is TPCK an example of irreversible inhibition?

It chemically modifies the enzyme, causing lasting inactivation under normal conditions.