1. Enzymes II
Dr. Kevin Ahern

2. E+S <=> ES <=> ES* <=> EP <=> E+P

3. E+S <=> ES <=> ES* <=> EP <=> E+P
Michaelis-Menten Kinetics
Rate of Formation
E+S <=> ES <=> ES* <=> EP <=> E+P

4. Product Substrate Enzyme Enzyme- Substrate Complex
Michaelis-Menten Kinetics
Substrate
Product
Enzyme
Enzyme-
Substrate
Complex

5. Substrate high Enzyme high ES low Product low
Michaelis-Menten Kinetics
Substrate high
Enzyme high
ES low
Product low

6. Kinetic Considerations
Pre-steady state
[E] and [ES]
varying widely
Steady state
[E] and [ES]
relatively constant

7. Pre-steady State Kinetics
Can give info on reaction mechanism, rate of ES formation

8. Enzymes
Kinetic Considerations

9. Enzymes High [S], High V0 (Enzymes Almost Always Busy)
Kinetic Considerations
Initial Velocity (V0) - Measured as [Product]/Time
Low [S], Low V0 (Enzymes Often Idle)
Substrate Concentration (Molarity)

10. Kinetic Considerations

11. Michaelis-Menten Kinetics
Parameters
Km = Substrate Concentration that Gives Vmax/2
NOT Vmax/2 Km

12. Michaelis-Menten Kinetics
Considerations
v0 =
Vmax[S]
Km + [S]
Enzymes That Don’t Follow Michaelis-Menten Kinetics Include Those That Bind Substrate Cooperatively - Binding of One Substrate Affects Binding of Others

13. Control of Enzyme Activity
Allosteric Control
Substrate Does Not Change Enzyme
Binding of Substrate
Substrate Does Change Enzyme
Binding of Substrate

14. Michaelis-Menten Equation
Vmax occurs when an enzyme is saturated by substrate
Vmax varies with amount of enzyme used
Km is a measure of an enzyme’s affinity for its substrate
Km value inversely related to affinity
High Km = Low Affinity
Low Km = High Affinity

15. Michaelis-Menten Kinetics
Vmax and Kcat
Vmax is Proportional to the Amount of Enzyme Used
in an Experiment - Not Useful for Comparing Enzymes
Since Vmax is a Velocity, and Velocity = [Product]/Time,
Vmax
[Enzyme Used] =
[Product]
[Enzyme Used] *Time
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 Kcat and
Does Not Vary with the Amount of Enzyme

16. Mutation leads to reduced Kcat/KM Diffusion of substrate limiting
Perfect Enzymes
Maximum Kcat/KM
Mutation leads to reduced Kcat/KM
Diffusion of substrate limiting

17. Triose Phosphate Isomerase

18. Enzyme Co-factors

19. 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/Km and 1/Vmax

20. Ribozymes

21. 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
V versus S
All of this working for you
Energy peaks
Are what an enzyme defeats
In its catalysis
Transition state
Is what an enzyme does great
And you should all know this
Catalytic action won't run wild - don't get hysteric
Cells can throttle pathways with an enzyme allosteric
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
Slow catalytically
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 do
Both axes are inverted then
You can determine Vmax and
Establish Km
for your
ENZYMES!
Sterically holding tight
Substrates positioned right
Inside the active site
Enzymes (Enzymes, enzymes, enzymes)
Metabolic Melody