Lec 1

Question 1

What is Kd?

Answer 1

the affinity of a protein/receptor for its ligand

Question 2

What Is Kcat?

Answer 2

rate of turnover of an enzyme of substrate -> P

Question 3

What is Km?

Answer 3

the affinity of a substrate for its enzyme - tells us the S conc needed to reach max rate

Question 4

Give 3 examples of proteins that can bind 2 or more ligands

Answer 4

• enzymes - kinases that bind ATP and protein
• ligand gated ion channels - some have a pentameric receptor site that can bind 5 ions
• haemoglobin - can bind 4 different oxygen molecules

Question 5

Draw a diagram and explain independence of binding 2 different ligands

Answer 5

• binding of one ligand at one site does not affect the binding of another ligand at another site
• can be binding sites that are close together or far apart
• same image for sites far apart

Question 6

Draw a diagram and explain +ve cooperativity of binding 2 different ligands

Answer 6

• electrostatic attraction promotes favourable interaction of 2 ligands at sites in close proximity

Question 7

Draw a diagram and explain -ve cooperativity of binding 2 different ligands

Answer 7

• electrosatic repulsion creates an unfavourbale interaction of 2 ligands that occupy sites in close proximity
• the interaction can be so unfavourable that it is extremely thermodynamically unfavourabel so this binding rarely happens

Question 8

Draw a diagram and explain competitive binding of 2 different ligands

Answer 8

• when 2 ligands bind at exactly the same site
• relies on the impossible fact that the 2 ligands can occupy the same site simulataneously
• also because most binding is reversible

Question 9

Give a defintion to describe ALLOSTERIC CONFORMATIONAL CHANGE

Answer 9

as proteins are not solid structures, more dynamic molecules, binding at one site can affect the binding of another site due to conformational changes in that site. this is without direct interactions between the 2 ligands

Question 10

What is the orthosteric site?

Answer 10

site which binds the main ligand / substrate binds

Question 11

Draw a diagram that outlines positive cooperativity via an allosteric change and describe it

Answer 11

• can see that binding at one site alters the structure of the other binding site & lowers the Kd of it for binding the other molecule
• eg to increase the strength of the interaction at the yellow site, the pink site needs to be occupied (top right)
• perfect interactions exist when both sites occupied

Question 12

Draw a diagram that outlines -ve cooperativity and explain it

Answer 12

• originally starts as both sites being complementary to their substrates and binding of one site alters the shape of the other binding site (less likely shape for binding)
• binding @ both sites result in unfavourable reactions
• binding @ one site will also increase the Kd for binding at the other site

Question 13

What is the equation to work out the fraction of ligand bound to protein?

Answer 13

fraction in PL = [L] / [L] + Kd

Question 14

Imaigine if we add a vast amount of M (saturating concn of M) and we want to determine what happens to the Kd. What happens to the Kd if binding is;

1. independent
2. +ve cooperativity
3. -ve cooperativity
4. competitive

Answer 14

1. Kd will be unaffected
2. Kd will be reduced
3. Kd increases
4. Kd increases substantially. fraction of protein bound to the other ligand will be 0

Question 15

Describe an experimental approach to investigate the cooperativity of binding (involves drawing graphs and adding in saturating concs)

Answer 15