Quantitative Analysis of Enzyme A ctivity Scott Sutherland Stony Brook University Steven Glynn Stony Brook University Lindsay Hinkle Harvard University Rosa Veguilla Harvard University Leon Dickson ID: 555249
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Group 1 – Interface of Chemistry and BiologyQuantitative Analysis of Enzyme Activity
Scott SutherlandStony Brook University
Steven GlynnStony Brook University
Lindsay HinkleHarvard University
Rosa VeguillaHarvard University
Leon DicksonHoward University
Kevin Jones
Howard UniversitySlide2
Goals and ObjectivesLearning 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 dataP
roduce the Michaelis-Menten plot from
experimental dataInterpret changes in reaction conditions from different
Michaelis-Menten
plots
Design
an experiment
to generate data for a
Michaelis-Menten
p
lotSlide3
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.Slide4
HIV-1 protease is crucial for the replication of HIVInhibiting 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)Slide5
Steady-state enzyme kinetics
Assumptions of Michaelis-Menten kinetics:
The reaction is at equilibriumThe reaction is at steady-stateSlide6
Choose the components of the HIV-1 protease reaction
HIV-1 protease
Viral polypeptide
HIV-1 protease/Viral polypeptide complex
Cleaved viral polypeptidesSlide7
An enzyme’s response to substrate can be visualized using the
Michaelis-Menten
plot
Michaelis-Menten Equation
Substrate concentration (
μM
)
Initial reaction velocity (
μ
M
sec
-1
)
V
max
K
M
V
max
/2Slide8
Activity 1Match the experimental data to the corresponding line on the plot of time-course reactionsRemember that the slope of the time-course corresponds to the rate of the reaction at a given substrate concentrationSlide9
Clicker question
Using your handout, identify which time-course corresponds to an initial [S] of 25 uM?Slide10
Activity 1IUse the reaction velocities from the time-course data to construct a Michaelis-Menten plot
Use your plot to estimate
Vmax and K
M for your enzymeSlide11
K
M
V
max
[S]VoSlide12
Clicker questionA. 0– 5 μMB. 8 – 12
μMC. 40 – 50 μMD. 80 – 100 μM
What value for KM did you determine from your
Michaelis-Menten plot?Slide13
Here’s what it should look like:Slide14
Is Group1avir a possible drug candidate against HIV?(intracelluar substrate concentration is ~20 μM
)
+ Group1avir
Using enzyme kinetics to evaluate drug candidates
V
max = 96.4 μM
K
M
= 10.2
μM
-
Group1avir
V
max
= 96.4
μM
K
M
= 47.0
μMSlide15
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 Slide16
In the next lab session you will:Measure rates of an enzyme-catalyzed reactionUse your data to construct a
Michaelis-Menten plot
Determine values for Vmax and K
MSlide17
Let’s remind ourselves what we’ve accomplishedIn 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