Unit 4 - Enzymes 2

Question 1

What are the two types of irreversible inhibition?

Answer 1

Affinity Labelling Agents or Reactive Substrate Analogues
- react directly with the enzyme active site

Mechanism Based Inhibitors or Suicide Inhibitors

• contain unreactive groups which are activated by the enzyme by chemical reaction and then react further with the enzyme active site to form covalent bonds to the enzyme.

Question 2

What is a Mechanism Based Inhibitor also known as?

Answer 2

Suicide Inhibitor

Question 3

What is a major cause of death in Europe and the USA?

Answer 3

Coronary Heart Disease

Question 4

What percentage of deaths due to heart disease in USA are due to atherosclerosis?

Answer 4

50%

Question 5

What is atherosclerosis?

Answer 5

Build up of fatty deposit on the inner walls of the arteries

Question 6

What is most of the deposits in atherosclerosis due to?

Answer 6

Cholesterol

Question 7

What is the rate limiting step of cholesterol synthesis?

Answer 7

Reduction of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonic acid
- HMGCoA reductase

Question 8

What was the first HIT compound that was screened when trying to find an inhibitor of HMGCoA reductase?

Answer 8

Mevastatin

Question 9

How does compactin or mevastatin work?

Answer 9

Mimics the susbtrate 3-hydroxy-3-methylglutaryl coenzyme A

- HMGCoA

Question 10

How do analogues of HMGCoA work?

Answer 10

Bind well to the enzyme active site as the enzyme stabilises these high energy intermediates as a way of increasing the rate of reaction

Question 11

What are statins?

Answer 11

competitive inhibitors of HMGCoA reductase

Question 12

How do statins bind HMGCoA?

Answer 12

Bind in two binding pockets in the enzyme active site

Question 13

Describe the binding sites of HMGCoA

Answer 13

Hydroxymethylglutaryl binding pocket

Hydrophobic binding pocket

Question 14

What is Ki?

Answer 14

Inhibition constant

Question 15

What is Km?

Answer 15

Michaelis constant

Question 16

Give some examples of currently used inhibitors of HMGCoA (statins)?

Answer 16

Atorvastatin
Fluvastatin
Pravastatin
Simvastatin

Question 17

Why are statins so popular?

Answer 17

Cardiovascular disease is a major healthcare burden in affluent countries
Increasing obesity and high cholesterol levels as major risk factors
Patients and healthcare providers willing to pay for effective brand medicines
Statins are orally bioavailable
- normally a one-a-day tablet
Widespread uses
- most patients with cardiovascular disease including elderly
- diabetes patients > 40 years
- preventative for asymptomatic individuals at increased risk

Question 18

How is atorvastatin metabolised?

Answer 18

Primary route of metabolism via CYP3A4 hydroxylation

Question 19

What are the side effects of atorvastatin?

Answer 19

Serious side effects are rare, not however class association with muscle effects

• myopathy
• myositis
• rhabdomyolysis

Statins can also alter liver function

• hepatitis
• jaundice

Question 20

Which enzyme gives rise to an increase in blood pressure?

Answer 20

Angiotensin converting enzyme, ACE

Question 21

How do angiotensin converting enzymes cause an increase in blood pressure?

Answer 21

3 mechanisms
- good target for control of blood pressure

Angiotensin 2 causes arteriolar vasoconstriction increasing blood pressure

constriction of the efferent arterioles of the kidney leads to increased perfusion pressure in the glomeruli.

Stimulation by ATII of the adrenal cortex to release aldosterone, a hormone that acts on kidney tubules, causes sodium and chloride ions retention and potassium excretion. Sodium is a “water-holding” ion, so water is also retained, which leads to increased blood volume, hence an increase in blood pressure.

Question 22

Where is angiotensinogen produced?

Answer 22

Liver

Question 23

How is angiotensinogen converted to angiotensin I?

Answer 23

Renin

Question 24

How is angiotensin I to angiotensin II?

Answer 24

Angiotensin converting enzyme, ACE

Question 25

How does angiotensin converting enzyme increase blood pressure?

Answer 25

Angiotensin II is a hormone and a potent vasoconstrictor that increases blood pressure
Angiotensin II causes release of the hormone aldosterone which stimulates sodium/water retention and also increase blood pressure
ACE catalyses hydrolysis of bradykinin
- a potent vasodilator
- increasing blood pressure

Question 26

What do proteases, such as ACE, usually have?

Answer 26

Binding pockets for each of the amino acids adjacent to the site of cleavage
N-terminal site are S1, S2, S3
C-terminal site are S1’, S2’, S3’

Question 27

What was known about ACE when work started to find an inhibitor?

Answer 27

The enzyme was known to use Zn2+ ions
A series of tripeptides were known to be substrate
- following sequence being optimal
- aromatic amino acid-Ala-Pro

Question 28

How does ACE work?

Answer 28

Cleaves the C-terminal 2 residues of the tripeptide substrate

Question 29

Why is sulphur used in ACE inhibitors?

Answer 29

Binds strongly to zinc

Question 30

What is the inhibitor constant, Ki, of captopril?

Answer 30

1. 7nM

- very potent

Question 31

What is the clinical use of captopril?

Answer 31

Effective on its own, 50%

Increased effectiveness with a diuretic, 90%

Question 32

What are the side effects of captopril?

Answer 32

Rash and loss of taste

- possibly due to presence of sulphur