- 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

- Use HIV-1 protease as a narrative framework. Briefly describe each of the steps of the HIV lifecycle and emphasize the role of HIV-1 protease. Explain that we need to understand enzyme kinetics to begin to design a drug against this enzyme.
- Recap briefly the general scheme of a enzyme catalyzed reaction. Have the students shout out how the components of the HIV-1 protease reaction fit into this scheme. What is the enzyme? What is the substrate? Etc.
- Point out that each point on the Michaelis-Menten plot corresponds to a reaction velocity determined from a time-course experiment. Walk them through Vmax and Km and be sure to explain how a low or high Km value impacts enzyme activity.
- Split students into groups of two and have them use the data from the bottom of the worksheet to determine which of the time-course slopes corresponds to a substrate concentration of 25 micromolar. Point out to students that the velocity is equal to dP/dT. This activity should take about 2 mins.
- Keep students in groups of two and have them use the data to construct a Michaelis-Menten plot. Point out that the [S] values for each time-course are now on the screen. This activity should take ~ 5 mins to construct the graph and 2 mins to estimate Vmax and Km. Students need to be prompted to finish on time.
- Clicker question to confirm that the students constructed their plots successfully.
- Compare the students plots to the calculated one.
- Tell them that we have developed a potential drug candidate against HIV-1 protease. We’re now going to use the effect of this drug on the kinetics of HIV-1 protease to evaluate if it’s a possible drug candidate. Have the students discuss among themselves if the drug “group1avir” may act as an inhibitor of the protease. Use this as a lead in to the discussion of the mechanisms of enzyme inhibitors in the following lecture. This activity should take ~2 mins to discuss and report back. Students should recognize that the KM increases and so the action of the enzyme has been impaired. This activity is a stepping stone to a broader discussion of what criteria is used to evaluate drug candidates. Point out that a drug does not need to inactivate an target to be effective. Potential discussion question. Why do we use drug cocktails for treatment rather than single drugs?
- Use this slide to illustrate that the a drug with a similar mode of action i.e. competitive inhibition was released onto the market and had a huge impact on the rate of death from HIV infection. Provides extra context about the exercise and helps students develop higher order thinking.
- Homework: