Enzyme Kinetics – Introduction

Enzyme Kinetics – Introduction

Steady-state conditions will be assumed to hold for the Michaelis-Menten (MM) Enzyme Kinetic Model.  This model requires the measurement of initial velocities (rate of reaction), conditions that happen when the substrate concentration is significantly in excess of that of the enzyme; consequently, product formation is minuscule and unimportant.  Thus one assumes the concentration of the substrate is essentially invariant during the period of data taking.  The same conditions and assumptions apply to a reversible inhibitor. 

For this exercise, you will demonstrate some of the major features of the MM model, including that velocity is directly proportional to the total enzyme concentration.  By collecting velocity data for a series of substrate concentrations you will obtain Km, the Michaelis constant, and Vm, the maximum velocity, for a given enzyme concentration. We will be using both the MM-plot and its linear derivative Lineweaver-Burke plot to determine both parameters.  Consult your textbook for these equations.

Enzyme Kinetics – Learning Objectives and Prelab Questions

Learning Objectives:

  1. Explore the effect of substrate concentrations on enzyme activity. 
  2. Use the substrate effects to explore Michaelis-Menten Kinetics 
  3. Plot MM and LB and determine Km and Vmax. 

Prelab Questions: 

  1. What assumptions are made in enzyme kinetics? 
  2. What is the Michaelis-Menten equation? 
  3. What is the Lineweaver-Burke equation

Enzyme Kinetics – Materials

  1. Spectrovis or Spectrovis plus set at Abs vs. time at 460 nm.
  2. One box of semi-micro disposable cuvettes.
  3. P10, P200, and P1000 pipets and tips.
  4. 2-3 scintillation vials

Reagents Needed:

  1. Horseradish peroxidase working solution
  2. 500 mL of 0.10M phosphate buffer, pH 7.0
  3. 200 ml of 9.8mM H2O2 (substrate one).
  4. 10% guaiacol solution in methyl alcohol (10 mL/100 mL MeOH), (substrate two)

Procedure:

  • On the spectrophotometer, set the data collection to ‘Absorbance vs. Time’, select a wavelength of 460 nm.  Under configuration, the collection should run at 1 datum/2sec, and run for 5 minutes/300 seconds.
  • In a clean set of cuvettes, prepare the following reaction mixtures (Table 3). Remember: Do not add the enzyme until you are ready to do the reaction.
TubeVol pH 7 bufferVol DyeVol WaterVol H2O2Enzyme
12.0 mL50 µL0.48 mL20 µL50 µL
22.0 mL50 µL0.45 mL50 µL50 µL
32.0 mL50 µL0.40 mL100 µL50 µL
42.0 mL50 µL0.30 mL200 µL50 µL
52.0 mL50 µL0.20 mL300 µL50 µL
62.0 mL50 µL0.10 mL400 µL50 µL
72.0 mL50 µL—-500 µL50 µL

Table 3

  • When ready, starting with cuvette #1, add 50 µL of the enzyme, stir and quickly place the cuvette in the spectrophotometer.
  • Collect for at least 5 minutes Remove the tube from the spectrophotometer and set aside in a beaker for disposal.
  • Save the run
  • Repeat with each of the tubes at a different substrate concentration and save all runs.
  • Export the data to excel, save it, and send the data to yourself for analysis. 

Enzyme Kinetics – Data Analysis

With the data collected, you will determine the relationship between the velocity (rate) of the reaction (Vo) and the substrate concentration (S) at the different substrate concentrations. To do this you have to convert your absorbance (OD) /min to concentration/minute. For this, the extinction coefficient of the guaiacol is 26.6 mM-1cm-1.

Note: Remember the Beer-Lambert law: OD = εcl

Where ε is the extinction coefficient,

c is the concentration and

l is the light path (here 1 cm).

So this can be set up neatly to be: c = (OD)/ εl.  

  • Plot the data as both Michaelis-Menten and Lineweaver-Burke graphs in excel. Calculate the Km and Vmax. Refer to Chapter 6 in Lehninger.   If you have questions about how to do this, then see the instructor.
  • The Michealis-Menten Plot is an example of a non-linear relation as such we will be using Solver from Excel to determine the Km and Vmax. 

Hints:

For the Michaelis-Menten Plot:  

  • The data points for your y-axis will be the slopes of your absorbance/time plots for the seven reactions (restrict your plot to the first 120 seconds/2 minutes).  The different slopes once converted to concentration (using Beer-Lambert Law) give you your y-axis data points, ie initial velocity (Vo). 
  • For your x-axis data points, we will be using the concentration of peroxide from the measured volume (1-7). Use m1v1=m2v2. The initial concentration of your peroxide is 9.8 mM. The initial volume will be on the table. The final volume = 2600 uL ( the total volume from each run on your table) and calculate for your final concentration.
  •  Plot the slope. (concentration/s) against the different peroxide, concentrations to obtain your MM plot.
  • The LB plot is a double reciprocal plot. 

Enzyme Kinetics – Post Lab Questions

  1. What Vmax and Km do you obtain from the Michaelis Menten plot?
  2. What Vmax and Km do you obtain from the Lineweaver-Burke plot?
  3. Are there any differences and state why the difference and which one will be more accurate?
  4. Look up Horseradish peroxidase on the Sigma-Aldrich website and discuss the following :
    1.  
      1. List some inhibitors of horseradish peroxidase
      1. What is the optimal pH for the enzyme
      1. Storage conditions
      1. Applications

Imagine finding the best recipe for your favorite dish and not being able to repeat it.  Hours of time wasted because of a lack of documentation. We do not want that to happen so for each lab there will be the submission of a lab report as the proof of our time in the lab. Each lab report should have the following:

  •  The sections listed in the table below. 
  • The report should either be typed out or written in Notability.  
  • The report should be submitted as a pdf before the next lab
SectionHeadingTimelineAdditional Information
1Title and date To be done before labWhat is the title of the lab and what  is the date of the experiment
2Purpose/ObjectivesTo be done before labWhat are the objectives of this lab?
3Prelab- QuestionsTo be done before labAnswer all questions  and prepare to discuss them in class
4Protocols (Materials and Methods)To be done before and during the labBefore lab – copy the  protocol into Notability During lab – Make a  special note of the changes and customizations that were  made during the experiment itself
5Results 1 – Data obtained directly during the experimentTo be done during the labData obtained during the lab. 
6Results 2 – Calculations, Images, Graphs, and tablesDepending on the time left after the experiment,  will be done during or after Graphs should not be a  screenshot but the actual graph copied from excel and pasted. Graphs should be labeled at each axes with units (if applicable). The graph should also have a title.  Images should be zoomed and cropped so that only salient data is present.  
7DiscussionAfter labAvoid discussing information  that was not presented in your results.  Summarize the results. State whether the results are expected or not.  Compare your data to previous work (if present) Your discussion should logically follow the results that were obtained. 
8Post lab QuestionsAfter labAnswer all the questions
Conclusion and Follow upAfter labState the significant  findings from the experiment Provide a future perspective on the work.
10Signature and Date of completionAfter lab
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