Lecture 8 Flashcards
E = S = P = ES = EP =
E = enzyme (active site) S = substrate P = product ES = enzyme-substrate complex EP = enzyme-product complex
- k =
- kcat =
- KM (Michaelis constant) = _______
- KD (dissociation constant) = _______
- *KM ≈ KD when ______
- k = rate constants for individual rxn steps
- kcat (turnover #; # of times each enzyme site converts substrate to product per unit time)
- KM (Michaelis constant) = (koff + kcat) / kon
- KD (dissociation constant) = koff / kon
* KM ≈ KD when kcat «_space;koff
When does KM ~= KD?
kcat «_space;koff
What are the 2 assumptions
- No product product inhibition
2. ES and EP do not leave the activate site therefore they’re not affected by concentration
What is the model that MM kinetics is based on?
E + S
—> k+1 / k-1 kcat
E + P
What is the Michaelis-Menten eqn and what does each symbol stand for?
Vo = Vmax[S] / KM + [S]
Vo = initial velocity Vmax = maximum velocity (limiting velocity) KM = Michaelis constant (= the [S] at Vmax/2) [S] = substrate conc
Draw the steady state assumption graph
L8 Slide 7
What is the pre steady state used for?
Sometimes used to measure individual rate constants
What are the 5 assumptions
- [E] «_space;[S]
- Initial rate of product formation can be measured before the substrate is DEPLETED and before product can INHIBIT the reaction - There is no product inhibition
- E + P to EP does not happen - There is no allostericity or cooperativity
- All active sites are independent of each other if the enzyme carries 2 or more active sites - It is an irreversible rxn
- EP to ES does not happen - [E] is constant
- Enzymes only act as catalysts therefore not consumed during the rxn
What are the purposes of enzyme kinetic experiments?
To determine the kcat, KM and kcat/KM
how do these values change depending on - Reaction conditions Or - Changes to the enzyme – mutations Or - Changes to the substrate Or - Changes to inhibitors (drugs) Etc…
Define kinetics
The measurement of reaction rates and their dependence on conditions
Slide 10
N/A
Draw a Michaelis-Menten kinetic curve
Slide 12
Describe Vmax. When is it always observed? What is it independent of?
- Maximum reaction rate
- It is always observed when
[S]»_space; [E] because enzyme is
saturated with substrate - It is CONSTANT and independent of further increases in substrate CONC
What is KM? What does it describe?
KM (≈KD)
Substrate concentration when
half of Vmax is achieved
Describes the affinity of the
enzyme for the substrate
What do Vmax and KM characterize?
- Enzyme
- Substrate
- Rxn conditions (e.g. Temp, pH, ionic strength)
Enzyme A:
kcat (s^-1) = 1000
KM (M) = 1000
kcat/KM (s^-1 x M^-1) = 1
Enzyme B:
kcat (s^-1) = 1
KM (M^-1) = 1
kcat/KM ( M^-1 x s^-1) = 1
Enzyme C:
kcat (s^-1) = 100
KM (M^-1) = 10
kcat/KM ( M^-1 x s^-1) = 10
Enzyme ___ has the highest conversion rate of the
substrate to product per active site per unit time
Enzyme ____ has the highest affinity for the substrate
Enzyme ____ is the most efficient enzyme overall
Catalytic efficiency is limited at ______
Enzyme A has the highest conversion rate of the
substrate to product per active site per unit time
Enzyme KM has the highest affinity for the substrate
Enzyme C is the most efficient enzyme overall
Catalytic efficiency is limited at 10^8 - 10^9 M^-1 x s^-1
Give an example of a highly efficient enzyme and its kcat/KM
Enzyme: B-lactamase Organism: E coli Substrate: Ampicillin kcat (s^-1): 1090 Km (M): 8 x10^-1 kcat/Km (M^-1 x s^-1): 8.7 x 10^8
What 3 things to consider when designing kinetic experiments
- Enzyme preparation
- Substrate design
- Rxn parameters
Slidd 16
N/A
Slide 17
N/A
What is FRET
Forster Resonance Energy Transfer
Describe FRET
N/A Slided 18
What are the rxn parameters?
- pH (Tris pH 8.0)
- Often between 7 to 8 - Ionic strength (100mM NaCl)
- Often between 50mM to 150mM - Additive (0.01% DDM)
- Based on protein (detergents, metal ions, co-factors) - Temperature (23°C)
- Most often done at 37°C - Others
- Types of reaction vessel (e.g. plastic, glass, quartz)
What does the Michaelis-Menten kinetic curve require?
- Requires at least 5 initial velocity points at
¼ KM, ½ KM, KM, 2KM, 4KM - “…10–12 data points should be collected, and they should evenly cover the range of 0.1–5 times Km, resulting in reaction velocities between 9 and 83 % of Vmax .
- Calculate error estimates for Vmax and Km
- General rule: Results without error estimates are almost useless. - Problem: KM is unknown
Solution: measure V0 at a wide range of [S]
Slide 22
N/A
Usually [E] ___% of [S]
5%
How to generate the Michaelis-Menten kinetic curve?
- Screen for the lowest [E] which still gives measurable activity.
- Screen a wide range of [S] to get a preliminary KM and hope that [E] is < 5% [S] at ¼ KM.
- Measure initial reaction velocity at ¼ KM, ½ KM, KM, 2KM, 4KM and make the Michaelis-Menten kinetic curve.
- Figure out Vmax.
Problem: What if [E] is > 5% [S] at ¼ KM?
? Optimize rxn parameters
Problem: How to extrapolate Vmax?
?
What is the linear form of MM eqn
Vo = Vmax x [S] / Km + [S]
=>
1/v = 1/Vmax + Km/Vmax x 1/[S]
Y = b + m X
Draw a Lineweaver-Burk plot
y-intercept = 1/Vmas x-intercept = -1/KM Slope = KM/Vmax
Why do we care about kinetic characterization of enzymes? THREE REASONS
- Compare different enzymes using the same substrate
- Evolution studies - Compare mutants of the same enzyme using the same substrate
- Understanding enzyme substrate interaction and enzymatic mechanism - Compare different substrates using the same enzyme
- Inhibitor design
Most highly catalytic efficient SPase I is found in what species
S. aureus
A single mutation increases ~ 44 times in catalytic efficiency for E. coli SPase I.
N/A
Compare the activity btwn lipo and non-lipo peptide substrates
Non-lipo-peptide:
Dabcyl-XXXXXXXXX(EDANS)X-NH2
Lipo-peptide: Dodecanoyl-X(Dabcyl)XXXXXXXXX(EDANS)X-NH2
Enzyme: Full length SpsB
The lipo-peptide has increased kcat by 1.85 times, kcat / Km by 50.9 times, and decreased Km by 27.5 times
Draw the rxn of a “one substrate” mechanism
E + S –>
What can experiments where u vary one substrate at a time allow you to determine?
Can help you determine the mechanism (order of binding and release) of multi-substrate reactions.
Draw random bi mechanism
2 substrates are required but doesn’t matter which one binds to enzyme first, then one product
Draw an ordered bi mechanism
2 substrates but specifically one binds first then substrate 2 can bind.
Draw a ping pong mechanism
1) 1st substrate binds to the enzyme,
2) transfers a functional group to the enzyme
3) Only then can the 2nd substrate bind and accept this fxnal group
Ping pong mechanism is a common mechanism for that type of enzyme
Transferases
Draw a random bi bi mechanism
two substrates and two products, which can be bound and released in any order.
Compare energy states using a graph of
1) Without catalyst
2) Lock and key
3) Induced fit
Slide 38
