Document d'enzymologie.

1. Group 1 – Interface of Chemistry and
Biology
Quantitative Analysis of Enzyme
Activity
Scott Sutherland
Stony Brook University
Steven Glynn
Stony Brook University
Lindsay Hinkle
Harvard University
Rosa Veguilla
Harvard University
Leon Dickson
Howard University
Kevin Jones
Howard University

2. Goals and Objectives
Learning Goal:
Students will have the ability to manipulate, interpret, and
produce visual representations of data describing kinetic
properties of enzymes
Learning Objectives:
Students will be able to:
• Determine reaction rates from experimental time-course
data
• Produce the Michaelis-Menten plot from experimental data
• Interpret changes in reaction conditions from different
Michaelis-Menten plots
• Design an experiment to generate data for a Michaelis-

3. Who you are:
Upper level Biochemistry major who has
completed Calculus and Introductory
Chemistry and Biology
We’re halfway through a lecture in
steady-state enzyme kinetics.
See tip sheet for topics you have
covered.

4. HIV-1 protease is crucial for the replication of
HIV
Inhibiting the activity of HIV-
I protease is a strategy for
combating the virus
The first step in designing an
inhibitor is to understand the
kinetic properties of the enzyme
(necessary for
HIV replication)

5. Steady-state enzyme kinetics
Assumptions of Michaelis-Menten kinetics:
1. The reaction is at equilibrium
2. The reaction is at steady-state

6. Choose the components of the
HIV-1 protease reaction
HIV-1 protease
Viral
polypeptide
HIV-1 protease/
Viral polypeptide
complex
Cleaved viral
polypeptides

7. An enzyme’s response to substrate can
be visualized using the Michaelis-Menten
plot
Michaelis-
Menten Equation
Substrate concentration
(μM)
Initial
reaction
velocity
(μM
sec
-1
)
Vmax
KM
Vmax/2

8. Activity 1
Match the experimental data to the
corresponding line on the plot of time-
course reactions
Remember that the slope of the time-course
corresponds to the rate of the reaction at a given
substrate concentration

9. Clicker question
Using your handout, identify which time-course corresponds to an
initial [S] of 25 uM?

10. Activity 1I
A. Use the reaction velocities from the
time-course data to construct a
Michaelis-Menten plot
B. Use your plot to estimate Vmax and
KM for your enzyme

11. KM
Vmax
[S]
Vo

12. Clicker question
A. 0– 5 μM
B. 8 – 12 μM
C. 40 – 50 μM
D. 80 – 100 μM
What value for KM did you determine from your Michaelis-
Menten plot?

13. Here’s what it should look like:

14. Is Group1avir a possible drug candidate against
HIV?
+ Group1avir
Using enzyme kinetics to evaluate drug candidates
Vmax = 96.4 μM
KM = 10.2 μM
-Group1avir
Vmax = 96.4 μM
KM = 47.0 μM

15. Trends in Annual Age-Adjusted* Rate of Death
Due to HIV Infection, United States, 1987−2009
Note: For comparison with data for 1999 and later years, data for 1987−1998 were modified to
account
for ICD-10 rules instead of ICD-9 rules.
*Standard: age distribution of 2000 US population
Saquinavir released onto
market by Roche

16. In the next lab session you will:
• Measure rates of an enzyme-
catalyzed reaction
• Use your data to construct a
Michaelis-Menten plot
• Determine values for Vmax and KM

17. Let’s remind ourselves what we’ve
accomplished
In this class you:
• Determined a reaction rate from experimental time-
course data
• Produced the Michaelis-Menten plot from experimental
data and estimate the kinetic parameters
• Used Michaelis-Menten plots to infer changes in
enzyme activity, e.g. in the context of a human disease