9 - Enzymes IV: Enzyme inhibitors

Question 1

Inhibitor

Answer 1

a molecule or ion that interacts with an enzyme and alters its structure to reduce its activity

• Important in the regulation of cellular activity
• Can use enzyme kinetics (Lineweaver-Burke) to investigate their modes of action

Question 2

2 types of inhibitor

Answer 2

reversible and irreversible

Question 3

Reversible inhibitors

Answer 3

(i) Competitive: active site e.g. ATP analogues (cancer) – kinase inhibition.
Need a low KM so can bind more effectively than substrate
(ii) Non-competitive: non-active site (allosteric) - change active site structure
(iii) Uncompetitive: binds ES complex

Question 4

Irreversible inhibitors

Answer 4

– Inhibitor binds permanently to enzyme. Usually mimics substrate to bind to catalytic centre
– Becomes covalently attached to catalytic group during catalysis -This step is irreversible for this type of inhibitor

Question 5

Irreversible inhibition; Aspirin

Answer 5

• Aspirin = Acetylsalicylic acid
– Salix alba - white willow
– Spiraea ulmaria – meadowsweet (herb)
• First marketed in 1899 (Bayer)
• Very commonly used member of the clas`s of drugs called NSAIDs 
(nonsteroidal anti-inflammatory drugs)
– Block prostaglandin synthesis (inflammatory response - blocked)
– cyclooxygenase

Question 6

Aspirin; An inhibitor of prostaglandin synthesis

Answer 6

• Enzyme = prostaglandin H2 synthase
– aka cyclooxygenase (Cox)
• Two major isoforms of cyclooxygenase;Cox1 and Cox2
– Cox1 widespread and constitutively expressed
– Cox2 released during inflammatory response
Aspirin targets both (non-selective)

Question 7

Aspirin; Cyclooxygenase inhibition

Answer 7

• Aspirin binds directly to active site: competes with arachidonate
• Nucleophilic attack of Ser-OH on acetyl side group of aspirin – serine is acetylated – loss of catalytic activity
• Aspirin – only member of the NSAID family that works by irreversible inhibition; others competitive inhibition (don’t donate an acetyl group to the serine)

Question 8

Aspirin; Prevention of blood clots

Answer 8

• Low dose -Prevention of thrombosis (blood clotting)
– Inhibits COX1 in blood platelets, preventing: Thromboxane A synthase

prostaglandin -> thromboxane -> aggregation = clotting

Question 9

Aspirin; Summary of pharmacological uses

Answer 9

• Normal dose -Anti-inflammatory and pain killer
– Inhibits COX1 and COX2 in most tissues, reducing inflammation, pain responses and fever
• Low dose -Prevention of thrombosis
– Inhibits COX1 in blood platelets, preventing prostaglandin/thromboxane-mediated aggregation
• Side effects -Stomach ulcers
– Prostaglandins prevent acid secretion (COX1)

Question 10

Irreversible inhibition; 5-Fluorouracil (5-FU)

Answer 10

• A chemotherapy drug that blocks DNA synthesis by inhibiting the production of pyrimidine bases (thymine and cytosine)
• 5-FU is the precursor for an irreversible inhibitor of thymidylate synthase
• Becomes covalently linked to the enzyme

Question 11

Kinetics of irreversible inhibition

Answer 11

k1 k2

E + S —> ES —> E + P

Question 12

Competitive inhibition

Answer 12

• Inhibitor competes directly with substrate for active site binding
• Enzyme can bind either the substrate or the inhibitor at the active site
• Inhibition can be overcome by adding more substrate - competitive
• Potency of inhibitor related to affinity for active site compared to substrate
• Competitive inhibitors are typically non-metabolizable substrate/ transition state analogues

Question 13

Competitive inhibition; Methotrexate

Answer 13

• 1948 - low folic acid (folate) diet associated with leukaemia remission
• Folate plays a role in DNA synthesis – could preventing this role treat cancer?
• Dihydrofolate reductase (DHFR) – involved in the utilisation of folate for DNA synthesis
• 1948 - Aminopterin, a folate analogue that inhibits DHFR identified as first chemotherapy drug
• A few years later, another folate analogue - methotrexate - is produced that binds more tightly and has better clinical effects

Question 14

Competitive inhibition; Methotrexate mode of action

Answer 14

• Inhibition of nucleoside base synthesis prevents DNA replication
• Prevents cell division
• Methotrexate is a major chemotherapy drug for treatment of cancers of the breast, bladder, head & neck, and lymphoma and lymphocytic leukaemias
• ‘Belt and braces’ – often used in combination with 5-FU to inhibit DNA synthesis via two different points (thymidylate synthase and DHFR)
• Inhibition of DHFR is effective but is not specific to cancer cells - affects all cells

Question 15

Competitive inhibition; Michaelis-Menten graph

Answer 15

• Vmax remains unchanged- If you add enough substrate, you can compete out the inhibitor completely
• KM increases with inhibitor- More inhibitor = lower affinity of active site for substrate so KM increase
• Slope of curve more shallow with inhibitor – velocity lower at a given [S] in the presence of inhibitor

Question 16

Competitive inhibition; Lineweaver-Burk plot

Answer 16

• Vmax remains unchanged- If you add enough substrate, you can compete out the inhibitor completely
• KM increases with inhibitor- More inhibitor = lower affinity of active site for substrate so KM increased
y intercept = 1/vmax

Question 17

Non-competitive inhibition

Answer 17

• Inhibitor binds in an allosteric fashion at a site other than the active site in the presence or absence of substrate (E or ES)
• Active site no longer able to catalyse conversion of S to P but can still bind substrate
• Very rare in nature – most inhibitors that act away from the active site tend to be irreversible inhibitors. Examples – heavy metals such as lead and mercury
• Overall effect on kinetics similar to irreversible inhibition

Question 18

Non-competitive inhibition; Michaelis-Menten graph

Answer 18

• Vmax reduced- Doesn’t matter how much substrate you add, you cannot compete out inhibitor so enzyme can never reach its full velocity potential in the presence of inhibitor
• KM unchanged- Affinity of the active site for the substrate not changed (not obscured by substrate); just the ability of the site to convert substrate to product
Shallower curve in the presence of inhibitor

Question 19

Non-competitive inhibition; Lineweaver-burk plot

Answer 19

• Vmax reduced (so 1/Vmax increases)- Doesn’t matter how much substrate you add, you cannot compete out inhibitor so enzyme can never reach its full velocity potential in the presence of inhibitor
• KM unchanged- Affinity of the active site for the substrate not changed (not obscured by substrate); just the ability of the site to convert substrate to product
Increase the point in which the line intercepts the y axis

Question 20

Mixed non-competitive inhibition;

Answer 20

• Both non-competitive and mixed non-competitive inhibitors bind allosterically
• Both can bind to either E or ES
• Non-competitive inhibitor does NOT alter binding of substrate (just catalytic ability of enzyme); Vmax decreased but KM unchanged
• Mixed inhibitor decreases ability of E to bind S; Vmax decreased and KM
increased (affinity decreased

Question 21

Mixed non-competitive inhibition Lineweaver-Burk plot

Answer 21

Mixed non-competitive inhibitors affect both aspects of active site – substrate binding and catalysis:

• Vmax decreased with inhibitor so 1/Vmax increases
• KM increases with inhibitor (affinity decreases) so x-axis intercept increases (becomes less negative)
• Result – lines intercept somewhere between Y- and X-axes

Question 22

Uncompetitive inhibition; aka anti-competitive

Answer 22

• Here, inhibitor binds only ES and not Enzyme
• Inhibitor binds proximal to active site – easiest to think of the inhibitor changing the active site of the enzyme such that a substrate molecule becomes ‘frozen’ into position:
– A. Bound substrate prevents other molecules getting in yet cannot be converted to P; Vmax decreased
– B. Bound substrate molecule is ‘frozen’ – increases apparent affinity of the active site for the substrate ; KM decreased
• Example: lithium as drug treatment for manic depression and bipolar disorder: Inhibitor of myo-inositol monophosphatase –IP3 signalling -mood

Question 23

Uncompetitive inhibition; Michaelis-Menten graph

Answer 23

• Vmax decreased- Substrate molecule ‘frozen’ – blocks others getting and yet cannot be converted to product
• KM decreased- Again – substrate molecule ‘locked in’ so apparent strength (affinity) with which the enzyme binds that molecule is increased

Question 24

Uncompetitive inhibition; Lineweaver-Burk plot

Answer 24

• Vmax decreased so 1/Vmax increases
• KM decreased so X-intercept becomes more negative
• End result – slope of line does not change
• Unlike competitive inhibition you cannot increase the reaction rate by increasing [S] – this will not ‘dislodge’ the inhibitor molecule; Vmax can never be reached in the presence of high [S]
Characteristic tramline pattern where the 2 lines never intercept with each other