E S ltgt ES ltgt ES ltgt EP ltgt E P MichaelisMenten Kinetics E S ltgt ES ltgt ES ltgt EP ltgt E P Rate of Formation Substrate Product Enzyme Enzyme ID: 688926
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Slide1
Enzymes II
Dr. Kevin AhernSlide2
E+S <=> ES <=> ES* <=> EP <=> E+PSlide3
Michaelis-Menten Kinetics
E+S <=> ES <=> ES* <=> EP <=> E+P
Rate of FormationSlide4
Substrate
Product
Enzyme
Enzyme-
Substrate
Complex
Michaelis-Menten KineticsSlide5
Michaelis-Menten Kinetics
Substrate high
Enzyme high
ES low
Product lowSlide6
Kinetic Considerations
Pre-steady state
[E] and [ES]
varying widely
Steady state
[E] and [ES]
relatively constantSlide7
Pre-steady State Kinetics
Can give info on reaction mechanism, rate of ES formationSlide8
Enzymes
Kinetic ConsiderationsSlide9
Enzymes
Kinetic Considerations
Initial Velocity (V
0
) - Measured as [Product]/Time
Low [S], Low V
0
(Enzymes Often Idle)
Substrate Concentration (Molarity)
High [S], High V
0
(Enzymes Almost Always Busy)Slide10
Kinetic ConsiderationsSlide11
Michaelis-Menten Kinetics
Parameters
K
m
K
m
= Substrate Concentration that Gives V
max
/2
NOT Vmax/2Slide12
Michaelis-Menten Kinetics
Considerations
v
0
=
V
max
[S]
K
m
+ [S]
Enzymes That Don’t Follow Michaelis-Menten Kinetics Include Those That Bind Substrate Cooperatively - Binding of One Substrate Affects Binding of Others Slide13
Control of Enzyme Activity
Allosteric Control
Substrate Does Not Change Enzyme
Binding of Substrate
Substrate Does Change Enzyme
Binding of SubstrateSlide14
Michaelis-Menten Equation
V
max
occurs when an enzyme is saturated by substrate
V
max
varies with amount of enzyme usedKm is a measure of an enzyme’s affinity for its substrateKm value inversely related to affinity
High K
m
= Low Affinity
Low K
m
= High AffinitySlide15
Michaelis-Menten Kinetics
V
max
and K
cat
V
max
[Enzyme Used]
=
[Product]
[Enzyme Used] *Time
V
max
is Proportional to the Amount of Enzyme Used
in an Experiment - Not Useful for Comparing Enzymes
The Two Concentrations Cancel Out.
The Result is a Number Per time (say 1000/second).
It is the Number of Product Molecules Made by Each Enzyme Molecule Per Time.
It is Also Known as the Turnover Number or K
cat
and
Does Not Vary with the Amount of Enzyme
Since V
max
is a Velocity, and Velocity = [Product]/Time,Slide16
Perfect Enzymes
Maximum K
cat
/K
MMutation leads to reduced Kcat/KMDiffusion of substrate limitingSlide17
Triose Phosphate IsomeraseSlide18
Enzyme Co-factorsSlide19
Michaelis-Menten Kinetics
Lineweaver Burk Plot
Also Called Double Reciprocal Plot
Uses Same Data as V vs. [S] Plot,
but Inverts All Data for the Plotting
Linear for Enzymes Following Michaelis Menten Kinetics
Direct Reading of -1/K
m
and 1/V
maxSlide20
RibozymesSlide21
Metabolic Melody
You know it's true
So when an effector fits
It will just rearrange
all the sub-u-nits
Inside an
ENZYME!
Flipping from R to T
ENZYME!
Slow catalytically
ENZYME!
No change in Delta G
You should relax
When seeking out the Vmax though
There are many steps
Enzymes
Lineweaver Burk Can save a scientist work
With just two intercepts Enzymes
Plotting all the data from kinetic exploration
Lets you match a line into a best fitting equation Here's what you doBoth axes are inverted then
You can determine Vmax and Establish Kmfor your ENZYMES!Sterically holding tight
ENZYMES!
Substrates positioned right
ENZYMES!
Inside the active site
Enzymes (Enzymes, enzymes, enzymes)
Enzymes
(To the tune of "
Downtown
")
Copyright
Kevin Ahern
Reactions alone
Could starve your cells to the bone
Thank God we all produce
Enzymes
Units arrange
To make the chemicals change
Because you always use
Enzymes
Sometimes mechanisms run like they are at the races
Witness the Kcat of the carbonic anhydrases
How do they work?
Inside of the active site
It just grabs onto a substrate
and squeezes it tight
In an
ENZYME!
CAT-al-y-sis
In an
ENZYME!
V versus S
In an
ENZYME!
All of this working for you
Energy peaks
Are what an enzyme defeats
In its catalysis
Enzymes
Transition state
Is what an enzyme does great
And you should all know this
Enzymes
Catalytic action won't run wild - don't get hysteric
Cells can throttle pathways with an enzyme allosteric