Enzyme-controlled reactions - The SSERC Team

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. 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 inhibition or

11. Non-competitive inhibition

12. Non-competitive inhibition

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

14. 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] Method

15. 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 Method (steps 1-5)

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

18. 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 I = Galactose (steps 6-7)

19. 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 I = Iodine (steps 8-9)

20. 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. Step 10

21. 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. Step 10

22. Results

23. Results