Recapping…
MBLG lecture 6
• Enzymes CANNOT change the
thermodynamics of a reaction: ∆Go or Keq
Dr. Dale Hancock
[email protected]
Lab 715 OR Room 377
• They CANNOT change the direction of a
reaction or the position of the equilibrium.
• They DO increase the rate of the reaction
by lowering the activation energy.
∆Go and Keq:
∆Go and Keq
Under standard conditions:
If the ∆Go is negative then each substrate molecule (
much more free energy than each product molecule (
<< 1 mole
Substrate
1 mole of
substrate
∆Go
>>1 mole
Product
Total free energy
∆Go and Keq
Reaction Specificity
Keq = [product]/[substrate]
= 20/3 or >1, hence the
equilibrium favours product
formation.
when the total free
energy difference, ∆G,
is zero
1 mole of
product
Total free energy
<< 1 mole
Substrate
At equilibrium,
) contains
).
>>1 mole
Product
• Enzymes have special active site pockets which
recognise only certain substrates.
• Enzymes are generally more specific than nonprotein catalysts.
• The active site is not exactly complementary to
the substrate, like a key in a lock. If it were, this
would stabilise the substrate making the
activation energy even higher.
• The active site is more complementary to the
transition state.
Total free energy
1
The Transition State
The Transition State
• An activated, transient state a very few
reactants reach
• Denoted by X#
• The reaction rate depends on [X#]
• Enzyme binds very tightly to the transition
state; much more tightly than to the
substrate.
X#, transition state
Free
Energy
(G)
Substrate
∆G0
Product
Progress of the reaction
The Transition State
X#
EX#
The enzyme binds
very tightly to the
transition state
Free
Energy
(G)
Reaction Rate Measurements
• The rate of a reaction is measured as the
#moles of product produced per unit time.
• The most user friendly units are µmol/min.
S
∆G0
P
Progress of the reaction
Measuring the rate of a reaction
• The term ASSAY is used in Biochemistry
to describe a reaction that measures
something; enzyme activity or the
concentration of a metabolite.
Measuring the rate of a reaction
The initial linear rate
Is used for all
enzyme kinetics
measurements
[product]
[product]
dP/dt
Time (min)
Time (min)
2
Reaction Rates can be
increased by:
The Effect of [substrate]
on a simple first order reaction without an enzyme
Increasing the
Slope = k, the rate constant
Reaction
rate
Temperature
[reactant]
A catalyst
[substrate]
The Effect of [substrate]
on a simple first order reaction without an enzyme
The Effect of [substrate]
on a simple first order reaction with an enzyme
Why is this graph so
different?
Slope = k, the rate constant
Reaction
rate
First order means the
reaction rate is
dependent on the
concentration of only
one reactant.
Reaction
rate
The non-enzyme
reaction
[substrate]
[substrate]
The Effect of [substrate]
The Effect of [substrate]
on a simple first order reaction with an enzyme
on a simple first order reaction with an enzyme
Something is saturated??
Reaction
rate
All the available enzyme is
saturated with substrate
Reaction
rate
[substrate]
[substrate]
3
Don’t confuse your graphs in the
exam!
The important parameters!
Vmax and KM
This graph is
used to
estimate the
rate of a
reaction
Vmax
This is a
time course!
[product]
This graph is
used to
estimate
Vmax and KM
Reaction
rate
The KM is the [S]
at ½Vmax
[substrate]
KM
Time (min)
The Effect of [substrate]
on a simple first order reaction with an enzyme
The Lineweaver-Burk Plot:
A double reciprocal plot used to find Vmax and KM
Vmax
The KM
describes the
shape of the
curve
Reaction
rate
1/Vmax
1/v
The KM is the [S]
at ½Vmax
KM
[substrate]
1/[S]
-1/KM
Note the difference in the relative sizes of
the substrate and enzyme!
Vmax: What is the maximum
speed the car can go at?
4
KM: how much petrol do you
The KM
need to travel at 60 kph?
Vmax
Two different isoenzymes
with different KMs for the
same substrate. Which has
the higher affinity for the
substrate?
Reaction
rate
Maybe the little
car is more
efficient?
KM
KM
The KM
[substrate]
The KM
Vmax
Vmax
Higher affinity because it
takes less substrate to attain
Vmax.
Reaction
rate
KM
KM
Higher affinity means a lower
KM
Reaction
rate
KM
[substrate]
KM
The KM and the Vmax
[substrate]
The KM
In most cases:
E+S
ES
KM
Measures the
affinity
of the enzyme
and substrate
E+P
KM =
Kcat =
Vmax/[E]
Measures how
fast the reaction
can go
Rate: ES
Rate: E + S
E+S
ES
OR
KM =
Rate of dissociation
Rate of association
5
The Significance of KM
Vmax and KCAT
• The [S] which gives ½ Vmax
• Vmax is [E] dependent.
• A measure of the affinity the enzyme has
for the substrate
• KCAT is the # molecules of substrate
converted to product per molecule of
enzyme in one second when the enzyme
is saturated with substrate (Vmax
conditions)
• A low KM means high affinity and vice
versa a high KM means low affinity
• The KM is independent of the [E]
• KCAT = Vmax/[E]
Vmax conditions
How we use Vmax
• If Vmax is enzyme dependent we can use
an assay operating under Vmax conditions
to measure the amount of enzyme present
in a sample.
• What are Vmax conditions?
• When the [S] is >10 X KM
Vmax
Reaction
rate
The KM is the [S]
at ½Vmax
KM
Vmax and [E]
This means that if
you add 2X as much
enzyme to the assay
you will have 2X the
rate of the reaction
Vmax
[enzyme]
[substrate]
10 X KM
Vmax and [E]
The Vmax is a
measure of the
amount of active
enzyme
Vmax
[enzyme]
6
Vmax
• Vmax is measured in Units (U).
• 1 Unit (U) is the amount of enzyme
required to release 1 µmole of product (P)
in 1 minute under Vmax conditions.
• You measure the rate of the reaction over
a short time (min).
Uses for the Vmax assay
• Whenever you need to measure the
amount of enzyme in a sample.
• In molecular biology and biochemistry; if
you want to see if an enzyme has been
switched on transcriptionally
• For diagnosis; elevated enzyme levels
denote tissue damage
Other uses for the enzyme
assay
• Often you use the enzyme to make
something; either a copy of a strand of
DNA or to digest a piece of DNA.
• Then you add lots of enzyme and run the
reaction to completion (often for 30 min to
1 h).
• You measure the final amount not the rate.
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