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MBB 324 > Lecture 8 > Flashcards

Lecture 8 Flashcards

1
Q 
E = 
S =
P =
ES =
EP =
A 
E = enzyme (active site)
S = substrate
P = product
ES = enzyme-substrate complex
EP = enzyme-product complex
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2
Q
  1. k =
  2. kcat =
  3. KM (Michaelis constant) = _______
  4. KD (dissociation constant) = _______
  5. *KM ≈ KD when ______
A
  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 &laquo_space;koff
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3
Q

When does KM ~= KD?

A

kcat &laquo_space;koff

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4
Q

What are the 2 assumptions

A
  1. No product product inhibition

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

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5
Q

What is the model that MM kinetics is based on?

A

E + S
—> k+1 / k-1 kcat
E + P

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6
Q

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

A

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

Vo = initial velocity
Vmax = maximum velocity (limiting velocity)
KM = Michaelis constant (= the [S] at Vmax/2)
[S] = substrate conc
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7
Q

Draw the steady state assumption graph

A

L8 Slide 7

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8
Q

What is the pre steady state used for?

A

Sometimes used to measure individual rate constants

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9
Q

What are the 5 assumptions

A
  1. [E] &laquo_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
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10
Q

What are the purposes of enzyme kinetic experiments?

A

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…
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11
Q

Define kinetics

A

The measurement of reaction rates and their dependence on conditions

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12
Q

Slide 10

A

N/A

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13
Q

Draw a Michaelis-Menten kinetic curve

A

Slide 12

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14
Q

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

A
  1. Maximum reaction rate
  2. It is always observed when
    [S]&raquo_space; [E] because enzyme is
    saturated with substrate
  3. It is CONSTANT and independent of further increases in substrate CONC
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15
Q

What is KM? What does it describe?

A

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

Describes the affinity of the
enzyme for the substrate

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16
Q

What do Vmax and KM characterize?

A
  1. Enzyme
  2. Substrate
  3. Rxn conditions (e.g. Temp, pH, ionic strength)
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17
Q

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 ______

A

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

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18
Q

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

A 
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
19
Q

What 3 things to consider when designing kinetic experiments

A
  1. Enzyme preparation
  2. Substrate design
  3. Rxn parameters
20
Q

Slidd 16

A

N/A

21
Q

Slide 17

A

N/A

22
Q

What is FRET

A

Forster Resonance Energy Transfer

23
Q

Describe FRET

A

N/A Slided 18

24
Q

What are the rxn parameters?

A
  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)
25
Q

What does the Michaelis-Menten kinetic curve require?

A
  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]
26
Q

Slide 22

A

N/A

27
Q

Usually [E] ___% of [S]

A

5%

28
Q

How to generate the Michaelis-Menten kinetic curve?

A
  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.
  4. Figure out Vmax.
29
Q

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

A

? Optimize rxn parameters

30
Q

Problem: How to extrapolate Vmax?

A

?

31
Q

What is the linear form of MM eqn

A

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

32
Q

Draw a Lineweaver-Burk plot

A 
y-intercept = 1/Vmas
x-intercept = -1/KM
Slope = KM/Vmax
33
Q

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

A
  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
34
Q

Most highly catalytic efficient SPase I is found in what species

A

S. aureus

35
Q

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

A

N/A

36
Q

Compare the activity btwn lipo and non-lipo peptide substrates

A

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

37
Q

Draw the rxn of a “one substrate” mechanism

A

E + S –>

38
Q

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

A

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

39
Q

Draw random bi mechanism

A

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

40
Q

Draw an ordered bi mechanism

A

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

41
Q

Draw a ping pong mechanism

A

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

42
Q

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

A

Transferases

43
Q

Draw a random bi bi mechanism

A

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

44
Q

Compare energy states using a graph of

1) Without catalyst
2) Lock and key
3) Induced fit

A

Slide 38

MBB 324 (17 decks)

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