Lecture 8

Question 1

E = 
S =
P =
ES =
EP =

Answer 1

E = enzyme (active site)
S = substrate
P = product
ES = enzyme-substrate complex
EP = enzyme-product complex

Question 2

1. k =
2. kcat =
3. KM (Michaelis constant) = _______
4. KD (dissociation constant) = _______
5. *KM ≈ KD when ______

Answer 2

1. k = rate constants for individual rxn steps
2. kcat (turnover #; # of times each enzyme site converts substrate to product per unit time)
3. KM (Michaelis constant) = (koff + kcat) / kon
4. KD (dissociation constant) = koff / kon
   * KM ≈ KD when kcat «_space;koff

Question 3

When does KM ~= KD?

Answer 3

kcat «_space;koff

Question 4

What are the 2 assumptions

Answer 4

1. No product product inhibition

2. ES and EP do not leave the activate site therefore they’re not affected by concentration

Question 5

What is the model that MM kinetics is based on?

Answer 5

E + S
—> k+1 / k-1 kcat
E + P

Question 6

What is the Michaelis-Menten eqn and what does each symbol stand for?

Answer 6

Vo = Vmax[S] / KM + [S]

Vo = initial velocity
Vmax = maximum velocity (limiting velocity)
KM = Michaelis constant (= the [S] at Vmax/2)
[S] = substrate conc

Question 7

Draw the steady state assumption graph

Answer 7

L8 Slide 7

Question 8

What is the pre steady state used for?

Answer 8

Sometimes used to measure individual rate constants

Question 9

What are the 5 assumptions

Answer 9

1. [E] «_space;[S]
   - Initial rate of product formation can be measured before the substrate is DEPLETED and before product can INHIBIT the reaction
2. There is no product inhibition
   - E + P to EP does not happen
3. There is no allostericity or cooperativity
   - All active sites are independent of each other if the enzyme carries 2 or more active sites
4. It is an irreversible rxn
   - EP to ES does not happen
5. [E] is constant
   - Enzymes only act as catalysts therefore not consumed during the rxn

Question 10

What are the purposes of enzyme kinetic experiments?

Answer 10

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…

Question 11

Define kinetics

Answer 11

The measurement of reaction rates and their dependence on conditions

Question 12

Slide 10

Answer 12

N/A

Question 13

Draw a Michaelis-Menten kinetic curve

Answer 13

Slide 12

Question 14

Describe Vmax. When is it always observed? What is it independent of?

Answer 14

1. Maximum reaction rate
2. It is always observed when
   [S]»_space; [E] because enzyme is
   saturated with substrate
3. It is CONSTANT and independent of further increases in substrate CONC

Question 15

What is KM? What does it describe?

Answer 15

KM (≈KD)
Substrate concentration when
half of Vmax is achieved

Describes the affinity of the
enzyme for the substrate

Question 16

What do Vmax and KM characterize?

Answer 16

1. Enzyme
2. Substrate
3. Rxn conditions (e.g. Temp, pH, ionic strength)

Question 17

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 ______

Answer 17

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

Question 18

Give an example of a highly efficient enzyme and its kcat/KM

Answer 18

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

Question 19

What 3 things to consider when designing kinetic experiments

Answer 19

1. Enzyme preparation
2. Substrate design
3. Rxn parameters

Question 20

Slidd 16

Answer 20

N/A

Question 21

Slide 17

Answer 21

N/A

Question 22

What is FRET

Answer 22

Forster Resonance Energy Transfer

Question 23

Describe FRET

Answer 23

N/A Slided 18

Question 24

What are the rxn parameters?

Answer 24

1. pH (Tris pH 8.0)
   - Often between 7 to 8
2. Ionic strength (100mM NaCl)
   - Often between 50mM to 150mM
3. Additive (0.01% DDM)
   - Based on protein (detergents, metal ions, co-factors)
4. Temperature (23°C)
   - Most often done at 37°C
5. Others
   - Types of reaction vessel (e.g. plastic, glass, quartz)

Question 25

What does the Michaelis-Menten kinetic curve require?

Answer 25

1. Requires at least 5 initial velocity points at ¼ KM, ½ KM, KM, 2KM, 4KM 2. “…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 . 3. Calculate error estimates for Vmax and Km - General rule: Results without error estimates are almost useless. 4. Problem: KM is unknown Solution: measure V0 at a wide range of [S]

Question 26

Slide 22

Answer 26

N/A

Question 27

Usually [E] ___% of [S]

Answer 27

5%

Question 28

How to generate the Michaelis-Menten kinetic curve?

Answer 28

1. Screen for the lowest [E] which still gives measurable activity. 2. Screen a wide range of [S] to get a preliminary KM and hope that [E] is < 5% [S] at ¼ KM. 3. Measure initial reaction velocity at ¼ KM, ½ KM, KM, 2KM, 4KM and make the Michaelis-Menten kinetic curve. 5. Figure out Vmax.

Question 29

Problem: What if [E] is > 5% [S] at ¼ KM?

Answer 29

? Optimize rxn parameters

Question 30

Problem: How to extrapolate Vmax?

Answer 30

?

Question 31

What is the linear form of MM eqn

Answer 31

Vo = Vmax x [S] / Km + [S] => 1/v = 1/Vmax + Km/Vmax x 1/[S] Y = b + m X

Question 32

Draw a Lineweaver-Burk plot

Answer 32

``` y-intercept = 1/Vmas x-intercept = -1/KM Slope = KM/Vmax ```

Question 33

Why do we care about kinetic characterization of enzymes? THREE REASONS

Answer 33

1. Compare different enzymes using the same substrate - Evolution studies 2. Compare mutants of the same enzyme using the same substrate - Understanding enzyme substrate interaction and enzymatic mechanism 3. Compare different substrates using the same enzyme - Inhibitor design

Question 34

Most highly catalytic efficient SPase I is found in what species

Answer 34

S. aureus

Question 35

A single mutation increases ~ 44 times in catalytic efficiency for E. coli SPase I.

Answer 35

N/A

Question 36

Compare the activity btwn lipo and non-lipo peptide substrates

Answer 36

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

Question 37

Draw the rxn of a "one substrate" mechanism

Answer 37

E + S -->

Question 38

What can experiments where u vary one substrate at a time allow you to determine?

Answer 38

Can help you determine the mechanism (order of binding and release) of multi-substrate reactions.

Question 39

Draw random bi mechanism

Answer 39

2 substrates are required but doesn’t matter which one binds to enzyme first, then one product

Question 40

Draw an ordered bi mechanism

Answer 40

2 substrates but specifically one binds first then substrate 2 can bind.

Question 41

Draw a ping pong mechanism

Answer 41

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

Question 42

Ping pong mechanism is a common mechanism for that type of enzyme

Answer 42

Transferases

Question 43

Draw a random bi bi mechanism

Answer 43

two substrates and two products, which can be bound and released in any order.

Question 44

Compare energy states using a graph of 1) Without catalyst 2) Lock and key 3) Induced fit

Answer 44

Slide 38