Enzymes- Kinetics
Dr Dawn Jones
Kinetics
• Kinetics is the study of how fast a reaction happens – the rate of the reaction
• Studying enzyme kinetics can help us understand the mechanism of the enzyme
• Many enzymes are regulated to control key reactions in the cell
Kinetics of simple reactions
A
k
B
For a reaction where A is converted to B, the rate or velocity (V) of A turning into B is:
V = k[A]
k is the rate constant, and determines how fast the reaction happens. The units of k are s-1
Kinetics of simple reactions
k
B
[A] (M)
A
Time (s)
As A gets used up the rate (V) slows down, since it is proportional to A
Enzyme-catalysed reactions
• The first step in an enzyme-catalysed reaction is the enzyme (E) binding the substrate (S)
• The substrate is then
converted into product
(P) and released
Enzyme-catalysed reactions
E+S
k1
k-1
ES
kcat
E+P
• kcat is the catalytic rate constant, sometimes called the turnover number
E+S
k1
k-1
ES
kcat
E+P
• The rate of making ES and breaking down ES often balance
• So [ES] is constant throughout the reaction
E+S
k1
k-1
ES
kcat
E+P
• Rate of formation of product is
V = kcat [ES]
• [ES] is constant
• Therefore V is constant (until the substrate starts to run out!)
• This is called steady state
Steady state enzyme kinetics
• Enzymes often work in steady state, at the start of the reaction
• Rate of formation of product (how many molecules are made per second) is
constant
What happens if you measure the rate (V) at different
concentrations of substrate (S)?
What happens if you measure the rate (V) at different
concentrations of substrate (S)?
•
Velocity (V) increases as substrate concentration [S]
increases
•
However, the graph levels off at a certain point
•
Adding more substrate no longer increases the rate
•
The velocity has reached a maximum and is saturated
Michaelis-Menton kinetics
• In 1913 Leonor Michaelis and Maud Menton proposed an equation to explain this
behaviour
• Known as the Michaelis-Menton equation
Maud Menton
Leonor Michaelis
[S]
V = Vmax
[S]+ K M
Vmax is the maximal velocity at saturating [S]
KM is the Michaelis constant
k−1 + kcat
KM =
k1
E+S
k1
k-1
ES
kcat
E+P
[S]
V = Vmax
[S]+ K M
What happens when the substrate concentration = KM?
[S] = KM
[S] = KM
[S]
V = Vmax
[S]+ K M
When [S] = KM, V = ½Vmax
KM is the concentration of substrate
that produces half the maximum rate
[S]
V = Vmax
[S]+ K M
Significance of KM
• KM is the [S] required for half maximum rate
• If KM is low – enzyme works fast at low [S]
• If KM is high – enzyme works fast only at very high [S]
An example
• Imagine an enzyme with a KM of 10 mM
• Assume Vmax = 100 µM/min
• Why do most enzymes have a KM close to the physiological concentration of their substrate?
[S] mM
10 mM
If KM = 10 mM If KM = 0.1 mM
50 µM/min
99 µM/min
5 mM
33 µM/min
98 µM/min
20 mM
67 µM/min
99.5 µM/min
• Enzyme activity is sensitive to changes in [S]
• Important in regulation of metabolism
Clinical insight- Alcohol intolerance
• Some people have an intolerance to drinking alcohol
• Causes a flushed (red) face after drinking alcohol
• Quite common in Asian population
• Caused by a deficiency in aldehyde dehydrogenase
(ALDH)
Clinical insight- Alcohol intolerance
• Alcohol (ethanol) is converted to acetaldehyde by alcohol dehydrogenase (ADH)
Ethanol+
NAD+
ADH
Acetaldehyde + NADH + H+
• Acetaldehyde is converted to acetate by aldehyde dehydrogenase (ALDH)
Acetaldehyde + NAD+ + H2O
ALDH
Acetate + NADH + 2H+
Clinical insight- Alcohol intolerance
• There are 2 forms of ALDH, one with low KM and one with high KM
• People with alcohol intolerance lack the low KM ALDH activity
• Only the enzyme with high KM is active
• This enzyme only starts to work when aldehyde concentrations are very high
• High levels of aldehyde in the blood cause symptoms