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

 -galactosidase

Competitive inhibition

OR

Non-competitive inhibition

Non-competitive Inhibition

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

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]

Method steps 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

Colorimeter R = ReferenceT = Test Direction of Beam

Cuvette no 20% galactose in buffer (cm 3 ) ONPG stock solution cm 3 ) buffer (cm 3 )* ONPG x 20 dilution (cm 3 ) A reading [ONPG] in cuvette x x x x x 10 -3

I = Galactose steps 6-7 Prepare cuvettes Each contains 2 cm 3 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 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min

I = Iodine steps 8-9 Prepare cuvettes Each contains 1 cm 3 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 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min

Step 10 Add buffer and diluted (x 20) ONPG to cuvette, mix and zero colorimeter Add 0.5 cm 3 diluted enzyme, start timer & invert cuvette, read in colorimeter after 2 min Compare with results after step 5.

Results

Results

Results