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Biochemistry > Enzyme kinetics > Flashcards

Enzyme kinetics Flashcards

Rate constant, Michaelis-Menten kinetics, week 7 practical, KM, Vmax and kcat.

1
Q

Which property asks ‘does a reaction proceed, how far’?

A

Thermodynamics.

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2
Q

Which property asks ‘how fast does a reaction proceed’?

A

Kinetics.

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3
Q

Give 2 reasons as to why the rate of a reaction is important.

A
  • Most cellular chemicals are
    thermodynamically unstable but rely on
    their rate of breakdown being too slow to
    matter (without catalysis).
  • Some diseases are caused by imbalances
    in the rates at which reactions are taking
    place.
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4
Q

What is k subscript un?

A

Rate constant for the unfolding of a protein/biomolecule under certain conditions.

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5
Q

Give the rate equation for the reaction A→C and give the order.

A

Rate = k[A]

First order

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6
Q

Give the rate equation for the reaction
A + B → C

A

Rate = k[A][B]

Second order

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7
Q

What is k subscript f?

A

Rate constant for forwards reaction.

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8
Q

What is k subscript r?

A

Rate constant for reverse reaction.

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9
Q

What does Michaelis-Menten kinetics assume?

(unsimplified reaction is on the summary sheet)

A

A simplified reaction scheme:
- Release of product is very fast (compared
to the reaction itself)
- Reverse reaction is sufficiently slow that
we can ignore it/the product is reacting
with something else so that it doesn’t
accumulate.
- The concentration of the enzyme-
substrate complex is at a steady state.

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10
Q

Give the simplified reaction scheme.

A

E + S –> k1, <–k-1 ES –> kcat E+P

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11
Q

What is the kcat?

A

The catalytic rate constant = the number of substrate molecules converted to product per enzyme molecule per unit time.

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12
Q

In 1913, the Michaelis-Menten equation was made due to simplified kinetics.
Give this equation.

A

V = Vmax[S]/(KM+[S])

V = reaction velocity
Vmax = maximal rate
[S] = substrate concentration
KM = Michaelis constant

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13
Q

What does the M-M equation tell you?

A

How rate varies with substrate concentration.

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14
Q

What is Vmax affected by?

A

The amount of enzyme but for a given amount of enzyme it is a constant.

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15
Q

How do you get the M-M curve?

(look at summary sheet)

A

Measuring rate at various substrate concentrations.

And if the reaction is following M-M kinetics.

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16
Q

Why is Vmax not usually reached?

A

It requires very high substrate concentration (needs to be almost infinitely high).

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17
Q

Therefore, what is the difficulty with M-M kinetics?

A

Finding Vmax in the first place.

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18
Q

Give an alternative.

(if confused, watch the recap)

A

Use different [S] and extrapolate to find out what Vmax must be.

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19
Q

How do you find KM?

A

Read off from Vmax/2 to x-axis.

20
Q

What is K subscript M?

A

The concentration of substrate at which half the active sites are filled.

Provides a measure of the substrate concentration required for significant catalysis to occur.

21
Q

KM provides an approximation of the substrate concentration in…

A

vivo.

22
Q

What is Vmax?

A

The maximum rate for the amount of enzyme used.

23
Q

If you double the amount of enzyme, Vmax…

A

doubles.

24
Q

What is k subscript cat?

A

The turnover number, the number of times each enzyme molecule goes through a catalytic cycle per second.

25
Q

kcat is independent of the amount of…

A

enzyme used in the reaction.

26
Q

Working out constants for a given enzyme requires several…

A

experiments.

Must measure the rate for at least 5 values of [S].

27
Q

For a good result, values of [S] should be on both sides of…

(look at graph on summary sheet)

A

KM.

The constants for the enzyme can then be calculated.

28
Q

What is the consequence of M-M equation?

A

When [S] drops so does the rate. In an experiment, the rate will often drop.

So record initial rate.

29
Q

Some experiments use a stopped assay.
What is this?
When is it okay?

A

Reaction is allowed to proceed for specific time until abruptly halted.

If rate is slow over observed period.

30
Q

What is the best way to calculate M-M constants?

A

Use a proper statistical package and non-linear regression to solve equation in laboratory.

31
Q

What is the second way to calculate M-M constants?

A

Rearrange the M-M equation to give a linear equation.

32
Q

Which 2 plots are used to calculate M-M constants?

A

Lineweaver-Burk plot.
Hanes-Woolf plot.

33
Q

Give the equation for a Lineweaver-Burk plot.

A

1/V = KM/Vmax x 1/[S] + 1/Vmax
y = mx + c

(reciprocal of M-M equation)

34
Q

What is the x-intercept of a Lineweaver-Burk plot?

A

-1/KM

35
Q

What are the issues with a Lineweaver-Burk plot?

A

Data points at high and low concentrations are weighted differently and therefore sensitive to errors.
- For low amounts of substrate, errors can creep in and get amplified due to reciprocal.

Not used in research laboratories due to variation.

36
Q

How do you get the Hanes-Woolf plot equation?
What is it?

A

Reciprocal M-M equation and then multiply both sides by [S].

[S]/V = 1/Vmax x [S] + KM/Vmax
y = mx + c

37
Q

What is the x-intercept?

A

-KM

38
Q

What does the Hanes-Woolf plot avoid?

A

1/problems

39
Q

When might kinetics differ from M-M (3)?

A

The enzyme is affected by the concentration of some other compound.
The reaction is more complicated than the simple scheme.
If the enzyme has multiple subunits and exhibits cooperativity.

40
Q

What is enzyme cooperativity?
What does it do to enzyme activity?

A

The binding of a substrate to one active site of a multi-subunit enzyme affects the binding affinity of substrate molecules to other active sites.

Enhances/diminishes enzyme activity.

41
Q

When [S]»>KM, the rate will be close to…

A

Vmax.

42
Q

But under normal conditions most enzymes are not…

A

saturated with substrate.

43
Q

Why is the rate constant kcat/KM generally a better measure of catalytic efficiency compared to just kcat?

(mathematical justification on summary sheet)

A

Because it takes into account both the rate of catalysis (kcat) and the formation of the ES complex (KM).

44
Q

What are diffusion limits (kcat/KM)?

A

Maximum rate enzymes work at.

45
Q

What do diffusion limits mean?

A

Rates cannot be higher than 10^8 and 10^9 S^-1M^-1.

Biochemistry (10 decks)

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