Binding Relationships and Km Flashcards
What is Kd in biochemistry?
Kd stands for the dissociation constant. It’s a measure of the affinity between two molecules, typically a ligand and a protein
What does it mean to have a lower Kd?
A lower Kd value indicates a higher affinity between the molecules. This means that they bind more tightly together, it takes a lower concentration of the ligand to bind to substrate
What are the units of Kd?
M (concentration)
What is the formula for Kd?
([L]* [P])/ [LP]
What are the two ways of getting a tighter binding ligand?
Need small Kd so either:
- Smaller K-1
- Larger K+1
K-1 is PL
K+1 is PL
Rank these ligands for protein X in order from the strongest to the weakest binding.
Lig 1, Kd = 10 mM
Lig2, Kd = 3 mM
Lig3, Kd = 3 µM
Lig4, Kd = 5 mM
Lig5, Kd = 30 mM
Lig6, Kd = 8 pM
Lig6 (Kd = 8 pM)
Lig3 (Kd = 3 µM)
Lig2 (Kd = 3 mM)
Lig4 (Kd = 5 mM)
Lig1 (Kd = 10 mM)
Lig5 (Kd = 30 mM): This has the highest Kd value, indicating the weakest binding affinity.
What are the ways of presenting the formula of Kd?
K off/K on
([P][L])/[PL]
(k-1)/(K+1)
What is fraction of bound protein?
This represents the proportion of protein molecules that are bound to a ligand at a given concentration
What is the formula for fraction of bound protein?
([PL]/ [P] Total) = ([L]/[L]+Kd)
How would you calculate [P] total?
[P] total = [P] + [PL]
How would you calculate [L] total?
[L] total = [L] + [PL]
What would happen if [P] total «[L] total?
This would mean [PL] «_space;[L] total
Imagine 10 chairs (protein binding sites) and 100 people (ligand molecules). You’ll have 10 people on seats ([PL]) but still have 90 people ([L] therefore 10 PL «_space;90 L
How would you calculate the [L] if you don’t know what it is?
[L] total = [L] + [PL]
1) Make [P] total «_space;[L] total
2) [PL] «_space;[L] total
3) Therefore [L] = [L] total
Do you want a larger or smaller K1 ([PL]) so that the ligand binds to the site quicker?
Want a large K1
Would you need a larger or smaller K-1 ([L][P]) for the ligand to leave the site slower?
You would need a smaller k-1 so the ligand leaves the binding site slower
What would go on the x axis and y axis of a binding curve?
X axis - [L]/ micromolars
Y axis - [PL]/[P] total
What would a suitable scale look like for the x and y axis on the binding curve?
X axis: 1-10
Y xais: 0-1
How would you calculate Kd from a binding curve?
Find half of the saturation point on the y axis and then found the corresponding number on the x axis
As we increase [S], rate increases at first but levels off at high [S]. Why? What does this tell us about the enzyme?
As you increase the substrate concentration ([S]), the reaction rate initially increases because there are more substrate molecules available to bind to the enzyme’s active sites.
However, at high substrate concentrations, all the enzyme’s active sites become saturated with substrate. This means that the enzyme is working at its maximum capacity, and adding more substrate won’t increase the reaction rate significantly.
What can we conclude if [E] is very low compared to [S]?
They’ll be very low [ES] and we can assume that [S] free = [S] total
What does the Michaelis Menten equation describe?
Describes the relationship between the initial rate of an enzyme-catalysed reaction and the concentration of the substrate
What is the equation for the Michaelis Menten equation?
Rate = kcat * [E] total * ([S]/[S]/+Km)
What is Kcat within the Michaelis-Menten equation?
Rate constant if all enzyme is in the ES complex
What is [E] total in the Michaelis-menten equation?
Total concentration of the enzyme, both free and bound to substrate
What is Km in the Michaelis-Menten equation?
Represents the substrate concentration at which the reaction rate is half-maximal
What is on the x and y axis of the Michaelis- Menten graph?
X axis - Substrate concentration
Y axis - Reaction velocity
How would you calculate Vmax without the graph?
Kcat (ES –> E + P) *[E] total
[E] how many enzymes available
Kcat is the maximum number of substrate molecules an enzyme can convert
What is the formula for rate of product formation using the Michaelis Menten equation?
Rate = (Kcat * [E] total * [ S]) / ([S] +KM)
