Enzyme kinetics Flashcards
Rate constant, Michaelis-Menten kinetics, week 7 practical, KM, Vmax and kcat.
Which property asks ‘does a reaction proceed, how far’?
Thermodynamics.
Which property asks ‘how fast does a reaction proceed’?
Kinetics.
Give 2 reasons as to why the rate of a reaction is important.
- 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.
What is k subscript un?
Rate constant for the unfolding of a protein/biomolecule under certain conditions.
Give the rate equation for the reaction A→C and give the order.
Rate = k[A]
First order
Give the rate equation for the reaction
A + B → C
Rate = k[A][B]
Second order
What is k subscript f?
Rate constant for forwards reaction.
What is k subscript r?
Rate constant for reverse reaction.
What does Michaelis-Menten kinetics assume?
(unsimplified reaction is on the summary sheet)
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.
Give the simplified reaction scheme.
E + S –> k1, <–k-1 ES –> kcat E+P
What is the kcat?
The catalytic rate constant = the number of substrate molecules converted to product per enzyme molecule per unit time.
In 1913, the Michaelis-Menten equation was made due to simplified kinetics.
Give this equation.
V = Vmax[S]/(KM+[S])
V = reaction velocity
Vmax = maximal rate
[S] = substrate concentration
KM = Michaelis constant
What does the M-M equation tell you?
How rate varies with substrate concentration.
What is Vmax affected by?
The amount of enzyme but for a given amount of enzyme it is a constant.
How do you get the M-M curve?
(look at summary sheet)
Measuring rate at various substrate concentrations.
And if the reaction is following M-M kinetics.
Why is Vmax not usually reached?
It requires very high substrate concentration (needs to be almost infinitely high).
Therefore, what is the difficulty with M-M kinetics?
Finding Vmax in the first place.
Give an alternative.
(if confused, watch the recap)
Use different [S] and extrapolate to find out what Vmax must be.
How do you find KM?
Read off from Vmax/2 to x-axis.
What is K subscript M?
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.
KM provides an approximation of the substrate concentration in…
vivo.
What is Vmax?
The maximum rate for the amount of enzyme used.
If you double the amount of enzyme, Vmax…
doubles.
What is k subscript cat?
The turnover number, the number of times each enzyme molecule goes through a catalytic cycle per second.
kcat is independent of the amount of…
enzyme used in the reaction.
Working out constants for a given enzyme requires several…
experiments.
Must measure the rate for at least 5 values of [S].
For a good result, values of [S] should be on both sides of…
(look at graph on summary sheet)
KM.
The constants for the enzyme can then be calculated.
What is the consequence of M-M equation?
When [S] drops so does the rate. In an experiment, the rate will often drop.
So record initial rate.
Some experiments use a stopped assay.
What is this?
When is it okay?
Reaction is allowed to proceed for specific time until abruptly halted.
If rate is slow over observed period.
What is the best way to calculate M-M constants?
Use a proper statistical package and non-linear regression to solve equation in laboratory.
What is the second way to calculate M-M constants?
Rearrange the M-M equation to give a linear equation.
Which 2 plots are used to calculate M-M constants?
Lineweaver-Burk plot.
Hanes-Woolf plot.
Give the equation for a Lineweaver-Burk plot.
1/V = KM/Vmax x 1/[S] + 1/Vmax
y = mx + c
(reciprocal of M-M equation)
What is the x-intercept of a Lineweaver-Burk plot?
-1/KM
What are the issues with a Lineweaver-Burk plot?
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.
How do you get the Hanes-Woolf plot equation?
What is it?
Reciprocal M-M equation and then multiply both sides by [S].
[S]/V = 1/Vmax x [S] + KM/Vmax
y = mx + c
What is the x-intercept?
-KM
What does the Hanes-Woolf plot avoid?
1/problems
When might kinetics differ from M-M (3)?
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.
What is enzyme cooperativity?
What does it do to enzyme activity?
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.
When [S]»>KM, the rate will be close to…
Vmax.
But under normal conditions most enzymes are not…
saturated with substrate.
Why is the rate constant kcat/KM generally a better measure of catalytic efficiency compared to just kcat?
(mathematical justification on summary sheet)
Because it takes into account both the rate of catalysis (kcat) and the formation of the ES complex (KM).
What are diffusion limits (kcat/KM)?
Maximum rate enzymes work at.
What do diffusion limits mean?
Rates cannot be higher than 10^8 and 10^9 S^-1M^-1.
