block 3 -enzymes Flashcards
ligand binding
Ligand binding (coming together of 2 molecules) is involved in almost all biochemical processes
e.g. structural components; enzymes; receptors/signalling; antibodies/immunity
→ protein:protein interactions; protein:ligand interactions; protein:DNA interactions; etc
-when the ligand conc increases the con of the protein ligand complex increases(hyperbolic relationship)
-equilibrium process and reversible
how is Kd calculated?
-Equilibrium Constant (KD): This value is key to understanding how strongly the ligand and protein bind together. It’s a ratio that compares the concentrations of the components at equilibrium.
KD = equilibrium dissociation constant = [P][L] / [PL]
[P] = concentration of the unbound protein.
[L] = concentration of the unbound ligand.
[PL] = concentration of the protein-ligand complex.
KD is essentially a measure of how much the system favors the dissociated state (P + L) versus the bound state (PL).
If KD < 1: The complex (PL) is favored, meaning the binding is strong, and the reaction is more likely to move towards forming the complex.
If KD > 1: The unbound proteins and ligands (P + L) are favored, meaning the binding is weaker, and the reaction will lean towards the reagents rather than forming the complex.
-when the conc of ligand= the kd = 50% binding
Gibbs free energy
The free energy (ΔG) represents the amount of energy available for doing work in a system.
A negative ΔG means the process is favorable and spontaneous (the reaction tends to move towards the formation of the complex).
A positive ΔG means the process is unfavorable and non-spontaneous (the reaction tends to move away from the formation of the complex).
equation to work out free energy
ΔG0 = -RT ln(KD):
ΔG0 is the change in free energy for the reaction to form the complex.
R is the gas constant (8.314 J/mol·K).
T is the temperature in Kelvin.
KD is the equilibrium dissociation constant, which helps determine how favorably the protein and ligand bind.
why is kd important to understand?
Measures Binding Affinity: KD quantifies how tightly a ligand (e.g., drug) binds to its protein target. Lower KD means stronger binding and higher affinity.
Drug Dosage Calculation: A lower KD value means a drug requires less quantity to achieve its effect (e.g., blocking 50% of binding sites).
Example: Drug A (KD = 1 mM) needs 4g to block 50% of binding sites, while Drug B (KD = 1 nM) needs only 4μg.
Biological Relevance: KD reflects the biological function:
Weak binding (higher KD) is useful for transient interactions, like leukocyte rolling in blood vessels (KD = 10⁻³ M).
Tight binding (lower KD) is crucial for stable interactions, such as antibodies binding to pathogens (KD = 10⁻¹⁰ M).
Predicts Drug Potency: Drugs with lower KD (tighter binding) require less drug to block 90% of binding sites, making them more potent.
Clinical Application: Helps in designing drugs with appropriate affinity for targets to achieve effective therapy with minimal dosage.
how do we measure kd?
-normally in a system you don’t now the conc of free proteins(P) but you know the total (PL)
-if you measure the fraction of protein bound you can determine the KD
-normally (L) is in large excess over (P) so we assume the total conc of the ligand is the same as free L conc
how do we work out the fraction of proteins bound?
(PL)/(p)total= (L)/kd+(L)
[PL]: concentration of the protein-ligand complex (bound protein)
[P]_{TOTAL}: total concentration of the protein, which is the sum of free protein and bound protein ([P] + [PL])
KD: the dissociation constant (a measure of binding affinity)
This equation essentially shows how the fraction of bound protein depends on the concentration of ligand (L) relative to the dissociation constant (KD).
when L = KD fraction of protein bound is 0.5 (50%)
when L = 10 KD ~0.9 (90%)
when L = 100 KD ~0.99 (99%)
when L = 0.1 KD 0.1 (10
methods to measure kd
-equilibrium methods= measure individual components [L], [P] and [PL]; or the fraction of protein bound at equilibrium e.g. Equilibrium dialysis, Analytical Ultracentrifugation, Isothermal titration calorimetry (ITC), Spectrophotometric titrations
-kinetic methods= measure reaction rates to determine association (kon) and
dissociation rate constants (koff) e.g.
- Rapid mixing techniques e.g. Stopped-flow fluorescence
- Surface plasmon resonance
isothermal titration calorimetry (ITC)
ITC = Isothermal Titration Calorimetry.
Measures heat change when a ligand binds to a protein.
Exothermic binding → releases heat.
Endothermic binding → absorbs heat.
Two identical cells:
Reference Cell = buffer only.
Sample Cell = buffer + protein.
The system keeps temperature constant (ΔT₁ = 0), and measures tiny heat changes needed to do that
How does isothermic titration calorimetry work?(ITC)
Add ligand step-by-step to protein.
Measure heat released or absorbed after each addition.
Plot the heat signal vs ligand concentration → fit the curve → extract:
KD (affinity)
ΔH (enthalpy change)
ΔG (free energy change)
Limitations of equilibrium methods
-Works well for weak to moderate binding (KD ~ mM to nM).=
when kd is very small (Very tight binding )(KD < nM):=
Hard to detect small changes accurately.so we use
Kinetic methods .
kinetics methods
measure reaction rates to determine association (kon) and
dissociation rate constants (koff) e.g.
- Rapid mixing techniques e.g. Stopped-flow fluorescence
- Surface plasmon resonance
kinetic methods equations
Protein + Ligand ⇌ Protein-Ligand Complex (PL)
Association rate = konx [P] x[L]
Dissociation rate = koff x[PL]
At equilibrium(rate of association is equal to the rate of dissociation)= KD = [P] × [L] / [PL] =KD = koff / kon
surface plasmon resonance
Measures changes in refractive index based on mass on a chip.
Sensor chip with immobilised protein, ligand flows over it.
When binding occurs → refractive index changes.
Measures kon and koff to determine KD:
KD = koff / kon
enzyme kinetics
Reaction:
E + S ⇌ ES → E + P
E= enzyme
S=substrate
ES= enzyme substrate complex
p= PRODUCET
Rate constants:
k1 = binding of enzyme and substrate
k-1 = dissociation of ES back to E + S
k2 = formation of product
Michaelis-Menten experiemnet
What are we trying to do?
We want to understand how the speed of an enzyme reaction (the rate) depends on how much substrate is present.
How is the experiment done?
You take a fixed amount of enzyme (E) — keep it the same for all tests.
You add different amounts of substrate (S) each time — starting from low amounts and increasing until you hit a plate and there is no more increase rate in reaction (zero order )
You measure how fast the reaction happens right at the beginning (the initial rate, v).
You plot a graph:
x-axis = substrate concentration [S]
y-axis = reaction rate v
What does the graph look like?
At low substrate: the rate increases quickly as you add more substrate.as enyzme con is in excess
At high substrate: the enzyme gets “full” (saturated), so even if you add more substrate, the rate levels off and stops increasing.
michaelis-menten equation
v = (Vmax × [S]) / (KM + [S])
Where:
v = rate of reaction
[S] = substrate concentration
Vmax = maximum reaction rate
KM = Michaelis constant(a measure of how good the enzyme is at binding S)=
E + S ⇌ ES → E + P
k1 = rate of E and S coming together
k-1 = rate of ES falling apart back into E and S
k2 = rate of ES becoming E + P (making product)
KM = (k-1 + k2) / k1
(It’s how easily the enzyme lets go of substrate compared to how easily it grabs it.)
k2 is often called kcat (“turnover number” — how fast one enzyme molecule makes product.)
km= Michaelis-Menten constant
When [S] = KM, the speed of the reaction is half of Vmax:
then: v= (vmax)/2
➔ KM tells you how much substrate you need to get the enzyme working at half its full speed.
If [S]»_space; KM, then:
v= vmax
➔ Meaning: when there’s lots of substrate, the enzyme works at full speed
important Assumptions for mechalis-menten
Steady-state assumption:
The amount of ES (enzyme + substrate complex) stays constant during the reaction (making and breaking ES happen at the same rate).
[S]0»_space; [E]0:
There is way more substrate than enzyme at the start.
Measure early:
Measure the reaction before too much product (P) is made.
➔ So the back-reaction (P going back to S) can be ignored.
Three Criteria for an Efficient Enzyme
1.Vmax should be large
➔ You want the enzyme to work as fast as possible.
➔ Vmax = [Total enzyme] × kcat
So if kcat is large (enzyme quickly turns substrate into product), Vmax will also be large.
2.KM should be small
➔ You want the enzyme to bind substrate strongly (needs less substrate to work well).
➔ Small KM = enzyme binds substrate easily.
- k2»_space; k-1
➔ Once the enzyme grabs the substrate, you want it to quickly make product, not just let go of the substrate.
➔ So you want k2 (forward reaction rate) to be much bigger than k-1 (unbinding rate).
But there’s a catch:
If you want k2 big (fast), and KM small (tight binding), there’s a compromise because k2 appears in KM (it affects both speed and binding).
You can’t have k2 both huge and tiny at the same time
how to measure catalytic efficiency
kcat/KM is a way to measure catalytic efficiency.
If it’s very high, the enzyme is nearly perfect (like acetylcholinesterase)
how does enzymes speed up the rate of a reaction?
hey help both physically and chemically:
Physical factors (Binding Help):
Hold the substrate in the right shape (stabilize transition state → not just the substrate).
Bring reacting groups close together.
Help line up the chemical groups perfectly (using charges, hydrophobic patches, etc.)
Chemical factors (Catalysis Help):
Provide chemical groups (like acids, bases) to assist breaking/making bonds.
Form temporary covalent bonds to split hard reactions into easier steps.
Stabilize extra charges that form during reaction.
Use metal ions (like Zn²⁺) to help reactions (metals can stabilize negative charges).
Use coenzymes (like vitamins) to carry chemical groups.
whats the difference betweeen thermodynamics and kinetics
theremodynamic= tells us if a reaction is energetically feasible
-kinetics= tells us about the reaction rate
the transition state theory in simpe terms
Imagine the reaction as a journey: you have to go over a hill (the transition state) to reach the other side (products).
The transition state is the highest energy point — the “top of the hill.”
Important facts:
The transition state is very unstable and exists only for about 10⁻¹³ seconds.
Transition state theory says that reactants and the transition state are in a kind of temporary equilibrium.
