Lecture 15 - Enzymes as drug targets

Question 1

The models of reversible enzyme inhibition make four assumptions. What are they?

Answer 1

1. binding of one molecule of inhibitor to the enzyme
2. inhibitor binding is reversible- covalent bonds not made
3. Linear kinetics will be observed- the rate of the reaction that we observe will not change except for depletion of substrate
4. Inhibitors are dead end – at high inhibitor concentrations there is no residual enzyme activity.

Question 2

Describe the structure of inhibitors- what can they resemble?

Answer 2

Resemble the substrate or the transition state.

Question 3

What values do inhibitors effect?

Answer 3

Km, Vmax or both

Question 4

How are inhibitors characterised?

Answer 4

By their Ki values

Question 5

Changes in Km and Vmax are related too…?

Answer 5

concentration of inhibitor and Ki value

Question 6

What is the Ki value?

Answer 6

inhibition constant which measures an inhibitors affinity for an enzyme

Question 7

What does a smaller Ki value mean? Relate answer to thermodynamic binding energy.

Answer 7

Smaller Ki indicates stronger inhibition, used to compare effectiveness of different inhibitors. Ki value is related to the thermodynamic binding energy- ΔG . When we have small Ki values we have tighter binding to the enzyme/higher potency

Question 8

Why is understanding the enzyme mechanism important for inhibitor development?

Answer 8

Many inhibitors resemble the transition state of the reaction.

Question 9

Name 4 types of reversible dead-end inhibitors.

Answer 9

competitive, non-competitive, mixed competitive, uncompetitive.

Question 10

How do competitive inhibitors work?

Answer 10

most common form.
Often arises because the inhibitor binds to the substrate binding site. Therefore, the inhibitor stops the substrate from binding.

Question 11

Sketch the Lineweaver burke plot for competitive inhibition. Describe effect on Km, Vmax and effect of substrate concentration increasing.

Answer 11

Km increases- x intercept shifts to left. A higher Km value means that more substrate is required to reach half Vmax- the enzyme has decreased affinity for its substrate due to competition with the inhibitor at the active site.

There is no change in Vmax- the y intercept is the same. This is because with enough substrate concentration, the reaction can still complete.

The amount of inhibition you get decreases when substrate concentration increases.

Question 12

Km (I) = Km (1 + [I]/Ki)
What do these values mean?

Answer 12

Km(I) – Km in the presence of the inhibitor
Km= Km in absence of inhibitor
[I] = inhibitor concentration
Ki = inhibition constant which measures an inhibitors affinity for an enzyme.

Question 13

What does a smaller Ki mean?

Answer 13

stronger inhibition.

Question 14

When [I] = Ki, what happens to the Km value?

Km (I) = Km (1 + [I]/Ki)

Answer 14

our Km value in the inhibited reaction will be twice that of the uninhibited because the fraction will reduce to 1+1

Question 15

What is non competitive inhibition? Is it common or uncommon?

Answer 15

Non-competitive inhibition involves the inhibitor binding to an area of the enzyme that is not the active site, causing a change in the shape of the enzyme which reduces its activity.

uncommon for single substrate enzymes.

Question 16

Sketch the Lineweaver Burke plot and describe non-competitive inhibition effects on Vmax, Km and effect of substrate concentration on inhibition.

Answer 16

Vmax is reduced- y intercept increases.
Km is not affected
Inhibition is not reduced by increasing substrate.

Question 17

What is a mixed competitive inhibitor? Are they common or not?

Answer 17

common form. Combination of competitive and non-competitive.

Question 18

Sketch a Lineweaver burke plot for mixed competitive inhibitors and describe effect on Km and Vmax

Answer 18

• Km increases as inhibitor concentration increases- moves to the left
• Vmax decreases (1/vmax increases)

Question 19

Is uncompetitive inhibition common? How does it work?

Answer 19

Uncommon. The inhibitor binds to the enzyme substrate complex. Prevents product formation.

Question 20

Describe effect of uncompetitive inhibition of Km and Vmax. How does changing substrate concentration effect inhibition?

Answer 20

Km and Vmax is reduced by the same amount- in other words, the substrate binds more tightly to the enzyme in the presence of the inhibitor.
Increasing substrate concentration increases inhibition. This is because the amount of enzyme in enzyme-substrate form will increase if there are more substrates. The inhibitor binds to enzyme substrate complexes, therefore inhibition increases.

Question 21

What do dose response curves measure?

Answer 21

drug potency at a fixed concentration

Question 22

How is a dose response curve plotted?

Answer 22

rate against log10 drug concentration.

Question 23

What is IC50 ?- dose response curves

Answer 23

Concentration of drug required for 50% of reduction in activity.

Question 24

What is the IC50 of this drug?

Answer 24

just above 30

Question 25

Tight binding inhibitors- when does it occur?

Answer 25

This occurs when the concentration of enzyme and inhibitor are similar. This means that the concentration of free inhibitor does not approximate to total inhibitor. The apparent Ki value will increase with increasing enzyme concentration.

Question 26

Why is tight binding inhibition desirable?

Answer 26

Tight binding inhibition is desirable because the enzyme inhibitor complex can have a very long half-life. You wont need to dose your patient very often. This means you get slow onset so patient might have to take medicine for a few days before they get full effect. This is often quite good. Low drug concentration means reduced potential for side effects.

Question 27

What do irreversible inhibitors do? give examples.

Answer 27

Irreversible inhibitors permanently inactivate the enzyme. Examples include penicillin and other B-lactam antibiotics. Enzymes inhibited are penicillin-binding proteins.