Enzymes 2

Question 1

Describe enzyme inhibitors

Answer 1

Inhibitors are any substances that can reduce enzyme activity by binding to the active site

An active site that is not covalently bound is a reversible inhibitor

• can be removed by dialysis
• characterised by dialysis

Question 2

How can enzyme inhibitors be classified

Answer 2

• Competitive
• Non-competitive
• Uncompetitive

Question 3

Describe reversible inhibition

Answer 3

This can be:

• Competitive inhibition
• Non-competitive inhibition

Question 4

Describe Competitive inhibition

Answer 4

Inhibitor competes with substrate at substrate-recognition site

• Inhibitors and substrates have close structural analogues (similar shapes)
• can therefore be overcome by increasing [substrate]

In the presence of a competitive inhibitor:

• Km is increased
• Vmax is unchanged

Question 5

What is Ki

Answer 5

Ki is the dissociation constant for inhibitor binding

SO:
- lower Ki means a stronger inhibitor

Question 6

Describe non-competitive inhibition

Answer 6

If an inhibitor does not compete with a substrate for its binding site
- termed non-competitive inhibition

They bind to a different site from the active site (allosteric site)

Non-competitive inhibitors:
- lower Vmax
(as they reduce the concentration of active enzymes, increasing [S} has no effect)
- Km is unchanged (substrate binding is unchanged)

Question 7

Describe some other types of inhibition

Answer 7

Mixed inhibition
- both Km and Vmax change with inhibitor bound

Uncompetitive inhibition
- both Km and Vmax fall

Substrate end product inhibition
- the kinetic effect depends on where the substrate or product bind (must be at a separate site, so NOT competitive, either non- or un-competitive or mixed)

Irreversible inhibition
- kinetic analyses don’t apply

Question 8

Describe irreversible inhibition

Answer 8

Some inhibitors bind to enzymes via covalent bonds (or very tightly):

• cannot be removed via dialysis
• reduced amount of available enzyme, can can only be overcome by synthesising more

e. g.CO attached to haem of haemoglobin
- lead forms covalent bond to protein cysteine (toxic)

Question 9

Describe suicide inhibitors

Answer 9

Substrate homologues
- they undergo part of the reaction cycle, generating a reactive intermediate that covalently attached to the enzyme

e. g.
- aspirin: irreversibly inhibits cyclooxygenase 1 + 2 (COX 1 and 2)
- Penicillin (irreversible inhibits bacterial transpeptidase)

Question 10

Describe allosteric regulation

Answer 10

Modulation of activity via reversible, non-covalent binding of small molecules
- enzyme activity is regulated in a way so they do not follow Michaelis-Menten kinetics

• this is the result of ‘effectors’ binding at allosteric sites, not the active or substrate-binding site
• binding of effectors changes the active-site conformation (can inhibit or potentiate)

Allosteric regulation is rapid, so is often the first response of cells to changes in conditions

Question 11

Describe multimers

Answer 11

Allosterically regulated enzymes are often composed of multiple subunits
- the allosteric site is often NOT on the catalytic subunit

The regulatory subunit cam also vary depending on the physiological conditions

Question 12

Describe the kinetics of allosteric regulation

Answer 12

Effectors display sigmoidal rather than hyperbolic kinetics
(i.e. distinct from standard inhibitors

K0.5 (the [Substrate] to give 50% of enzyme sat) is used instead of Km=

Allosteric activators and inhibitors can affect K0.5 and/or Vmax

Question 13

Give some advantages of allosteric regulation

Answer 13

• Can both activate or inhibit enzymes
• Effectors need not resemble substrate/product
• Regulation is rapid

Question 14

Describe allosteric activators/positive effectors (used interchangeably)

Answer 14

Effectors that increase enzyme activity

Question 15

Describe allosteric inhibitors/negative effectors

Answer 15

Effectors that decrease enzyme activity

Question 16

Describe cooperativity (in relation to substrate, enzyme, multimer)

Answer 16

When a substrate binds on a subunit in a multimer, it can affect the catalytic properties of the other subunits

• if catalysis is enchanted, this is positive cooperativity
• if catalytic activity is reduced, this is negative cooperativity

Question 17

Describe ‘The concerted model of enzyme cooperativity

Answer 17

Enzyme subunits adopt 2 different conformations:

• T (taut)
• R (relaxed)

Subunits start in the low affinity ‘T’ conformation, so substrate binds poorly

Binding depends on equilibrium between T and R states
- Binding of the first substrate molecules stabilises the high affinity ‘R’ state

Activators act by increasing the fraction of the enzyme un the ‘R’ state

Question 18

Describe the ‘Sequential’ model of enzyme cooperativity

Answer 18

Subunits convert to the R state on substrate binding, increasing the remaining subunits substrate affinity
- The sequential model can explain negative cooperativity (which the concerted model cannot)

In this model, inhibitors stabilise the T state, and activators stabilise the R state