1. Enzyme-controlled reactions
The SSERC Team

2. Metabolism and Survival
Metabolism is essential for life
Suggested learning activities include:
Enzyme induction experiments such as ONPG and lactose metabolism in E. Coli and
p-Glo experiments
Experiments on reaction rates with increasing [S]

3. Metabolism and Survival
Metabolism is essential for life
Suggested learning activities...
Investigate inhibition of
b-galactosidase by galactose and its reversal by increasing [ONPG]
Experiments on product inhibition with phosphatase
Experiments on ATP dependent reactions e.g. luciferase, luminescent reactions

4. Human Cells Cell metabolism Suggested learning activities include:
Enzyme induction experiments such as ONPG and lactose metabolism in E. Coli and
p-Glo experiments
Experiments on reaction rates with increasing [S]

5. Human Cells Cell metabolism Suggested learning activities...
Investigate inhibition of
b-galactosidase by galactose and its reversal by increasing [ONPG]
Experiments on product inhibition with phosphatase
Experiments on ATP dependent reactions e.g. luciferase, luminescent reactions

6. Paul Beaumont / Kate Andrews Kath Crawford / Lorraine Bruce
Effect of competitive and non–competitive inhibitors on ‑galactosidase
Paul Beaumont / Kate Andrews
Kath Crawford / Lorraine Bruce

7. b-galactosidase

8. b-galactosidase

9. Competitive inhibition

10. Competitive inhibitionor

11. Non-competitive inhibition

12. Non-competitive inhibition

13. Effect of [substrate] Competitive inhibitor Non-competitive inhibitor
Increasing [substrate] displaces inhibitor from active site
Non-competitive inhibitor
Increasing [substrate] has little or no effect on enzyme activity

14. Method Carry out reaction
Without inhibitor at low [S] (ONPG)
In presence of inhibitor
Galactose
Iodine
[I] chosen to completely inhibit reaction and look at increasing [S]

15. Method (steps 1-5) Dilute β-galactosidase (enzyme)
Dilute stock ONPG (substrate)
Mix diluted buffer and diluted ONPG in test tube, zero colorimeter
Add diluted enzyme
Read absorbance after two minutes

16. Colorimeter
Direction
of Beam
R = Reference T = Test

17. 1 2 - 1.0 0.25 0.75 3 0.5 4 5 Cuvette No 20% galactose in buffer (cm3)
ONPG stock solution (cm3)
Buffer
(cm3)
ONPG x 20 dilution
Absorbance 1 2 -
1.0
0.25
0.75 3
0.5 4 5

18. I = Galactose (steps 6-7) Prepare test tubes
Each tube contains 2 cm3 galactose (I)
Tubes 1 – 5 contain increasing [S]
NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 5
Mix, place in cuvette  colorimeter, reference colorimeter
Return to test tube
Add 0.5 cm3 diluted enzyme, start timer & mix, add to cuvette, read Absorbance after 2 min

19. I = Iodine (steps 8-9) Prepare test tubes
Each tube contains 1 cm3 iodine (I)
Tubes 1 – 4 contain increasing [S]
NOTE use ONPG stock and buffer for all 4 tubes
Mix, place in cuvette  colorimeter, reference colorimeter
Return to test tube
Add 0.5 cm3 diluted enzyme, start timer & mix, add to cuvette, read Absorbance after 2 min

20. Step 10
Add buffer and diluted (x 20) ONPG to test tube, mix and reference colorimeter
Add 0.5 cm3 diluted enzyme, start timer, mix read in colorimeter after 2 min
Compare with results after step 5.

21. Step 10
Mix diluted buffer and diluted ONPG in test tube, zero colorimeter
Add diluted enzyme
Read absorbance after two minutes
Compare with results after step 5.

22. Results

23. Results