1. Lab 6: Enzyme Catalysis

2. Purpose of the lab exercises:
Review the function and characteristics of enzymes
Become familiar with the spectrophotometer
Determine the optimal reaction conditions for the enzyme peroxidase

3. Chemical Reactions Involves breaking and making bonds
Requires an exchange of energy between the molecules and the environment
The energy required to start a reaction: ACTIVATION ENERGY (EA)
Energy required to break the chemical bonds in the reactants

4. Enzymes: Activation Energy
Decrease the activation energy of a reaction
Activation Energy:
Energy required to break bonds in reactants
Lowering activation energy speeds up reaction

5. Enzymes: Optimal Conditions
Each enzyme has an optimal set of conditions at which the maximum reaction rate occurs
Enzymatic reactions are affected by:
e.g.: Heat-tolerant bacteria enzymes optimally function at higher temperatures than most enzymes in humans
Temperature pH
Enzyme conc.
Substrate conc.

6. Chemical Reactions & Enzymes
BUT, these things cannot be done in a cell
The, internal and external environment of a cell is relatively constant
So, how does a cell proceed with reactions?
Enzymes!!
Catalytic proteins
Change the rate of a chemical reaction
Not consumed by the reaction

7. How Enzymes Works
The substrate (reactant) moves toward the enzyme’s active site
The chemical reaction is triggered by the enzyme
The enzyme releases the product

8. Enzymes
Are very specific for their substrate

9. Enzymes: Catalytic Cycle

10. Carrying out 4 separate experiments varying:
Enzymes: Peroxidase
Today’s lab: Determine optimal conditions for the enzyme peroxidase
HOW?
Carrying out 4 separate experiments varying:
TEMPERATURE pH
Substrate used in experiments: Mixture of guaiacol (resin from a small tree) + hydrogen peroxide

11. 2,2’ dihydroxy-3,3’ dimethoxybiphenyl (colorless)
Peroxidase
H3CO OH
OCH3
Guaiacol
(Yellow) +
H2O2
Radical (unstable)
2 H2O
2,2’ dihydroxy-3,3’ dimethoxybiphenyl (colorless) O
3,3’-dimethyoxybipheno-4,4’ quinone
(Red)
Peroxidase
Catalyzes oxidation of guaiacol Transfers hydrogen and electrons from guaiacol to hydrogen peroxide
Color change (substrate is yellow and product is red)
Monitor progress using a spectrophotometer

12. Spectrophotometer
Measures the amount of light passing through a solution at a specific wavelength
Determines how much light was absorbed by a solution

13. Spectrophotometer In today’s experiments, as the reaction progresses:
More and more reddish product forms
Absorbance readings will increase

14. Experiment 1 (Temperature)
Enzyme Experiments
Four students (two pairs) in each row should work together
One pair does Experiments 1 (temperature)
The other pair does Experiments 2 (pH)
Experiment 1 (Temperature)
Experiment 2 (pH)

15. Lab Summary
Need a graph of A500 as a function of time for the temperature experiment
Steps:
Mark the temperature for each of the four curves in a legend to the graph
Calculate from the initial region (straight line portion) of each curve the rate of the reaction at each temperature (A500 per second) and enter these values next to each curve

16. Lab Summary “Graphing”
Mark the temperature clearly for each of the four curves or in a legend to the graph

17. Lab Summary “Graphing”
Calculate rate from the initial region (straight line portion) of each curve at each temperature (A500 per second) and enter these values next to each curve
0.002 A500/sec

18. Lab Summary “Graphing”
The rate of the reaction is linear for about 20 seconds, (straight-line portion” of the reaction) After ~ 20 seconds: There are not enough substrate molecules So the rate slows down
Calculate the rate of reaction using only the straight-line portion of the reaction data

19. Lab Summary “Calculating Rate”
Rise
Run
Rise
X2 - X1
Y2 - Y1 =
0.035 A500
Rate =
0.035 A500
15 sec
Run
15 sec
=0.002 A500/sec

20. Lab Summary
In a similar way, the rate of reaction needs to be calculated for 25oC, 35oC, 45oC and 55oC
Temp oC Rate, A500/sec
On a separate set of coordinate axes, make a second graph relating reaction rate to temperature
Label the optimal temperature (the temperature at which the enzyme exhibits its highest rate of activity)

21. Lab Summary “Graphing”
The optimal temperature is 35OC
Temp oC Rate, A500/sec

22. Lab Summary “Conclusion”
At 25oC
Molecules are moving more slowly
Substrate collides less frequently with enzyme
Slower rate of reaction
At 45 & 550C The enzyme becomes “denatured”
The enzyme no longer functions properly

23. 500
60 sec

24. A500/sec

25. Lab Summary
Similar analysis of data for the pH experiments should be done

26. Start with 0 transmittance
filter
Make sure level set to nm
Start with 0 transmittance
Change from transmittance (0) to absorbance on mode (19999)
Set wavelength to 500 nm
For blank set absorbance to 0

27. So, let’s get to it!