Effect of competitive and non – competitive inhibitors on  ‑ galactosidase

1. Effect of competitive and non–competitive inhibitors on ‑galactosidase Paul Beaumont/Kath Crawford Gordon Moore/Anne Adams Science & Plants for Schools

2. b-galactosidase

3. b-galactosidase

4. Competitive inhibition

5. Competitive inhibition OR

6. Non-competitive inhibition

7. Non-competitive Inhibition

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

9. 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]

10. Methodsteps 1-5 • Dilute β-galactosidase (enzyme) • Dilute stock ONPG (substrate) • Mix diluted buffer and diluted ONPG in cuvette, zero colorimeter • Add diluted enzyme to cuvette • Read absorbance after two minutes

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

13. I = Galactosesteps 6-7 • Prepare cuvettes • Each contains 2 cm3 galactose (I) • Cuvettes 1 – 6 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 6 • Mix, cuvette in colorimeter, zero colorimeter • Add 0.5 cm3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min

14. I = Iodinesteps 8-9 • Prepare cuvettes • Each contains 1 cm3 iodine (I) • Cuvettes 1 – 3 contain increasing [S] NOTE – diluted (x 20) ONPG into tube 1, ONPG stock and buffer into tubes 2 – 3 • Mix, cuvette in colorimeter, zero colorimeter • Add 0.5 cm3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min

15. Step 10 • Add buffer and diluted (x 20) ONPG to cuvette, mix and zero colorimeter • Add 0.5 cm3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min • Compare with results after step 5.

16. Results

17. Results

18. Results