Effect of competitive and non–competitive inhibitors on ‑galactosidase Paul Beaumont/Kath Crawford Gordon Moore/Anne Adams Science & Plants for Schools
Apr 01, 2015
Effect of competitive and non–competitive inhibitors on ‑galactosidase
Paul Beaumont/Kath CrawfordGordon Moore/Anne Adams
Science & Plants for Schools
-galactosidase-galactosidase
O
OHH
H
HOCH2
OH H
HO
H
O
OHH
H
HOCH2
OH H
H H
H
O
OH
Lactose
-galactosidase
O
OHH
H
HOCH2
OH H
HO
H
OH
H
Galactose
O
OHH
H
HOCH2
OH H
H
HO
H
OHGlucose
+
-galactosidase-galactosidase
-galactosidase
O
OHH
H
HOCH2
OH H
HO
H
OH
H
GalactoseO
OHH
H
HOCH2
OH H
HO
H
O
NO2
ONPG(2-nitrophenyl - -D-galactopyranoside)
+HO
NO2
2-nitrophenol(o-nitrophenol)
Competitive Competitive inhibitioninhibition
Enzyme active site
Substrate Inhibitor
Competitive Competitive inhibitioninhibition
SubstrateInhibitor
OR
Non-competitive Non-competitive inhibitioninhibition
Enzyme active site
Regulator site
Inhibitor
Substrate
Non-competitive Non-competitive InhibitionInhibition
+
Effect of [substrate]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
MethodMethodCarry out reaction
– Without inhibitor at low [S] (ONPG)– In presence of inhibitor
GalactoseIodine
[I] chosen to completely inhibit reaction and look at increasing [S]
MethodMethodsteps 1-5steps 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
ColorimeterColorimeter
R = Reference T = Test
Direction of Beam
Cuvette no
20% galactose in buffer
(cm3)
ONPG stock
solution cm3)
buffer (cm3) * ONPG x 20 dilution
(cm3)
A reading
[ONPG] in cuvette
1 2 - - 1.0 4.3 x 10-4
2 2 0.25 0.75 - 2.15 x 10-3
3 2 0.5 0.5 - 4.3 x 10-3
4 2 0.75 0.25 - 6.45 x 10-3
5 2 1.0 0 - 8.6 x 10-3
I = GalactoseI = Galactosesteps 6-7steps 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
I = IodineI = Iodinesteps 8-9steps 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
Step 10Step 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.
ResultsResults
-galactosidase 24 Jan 2006
0
0.1
0.2
0.3
0.4
0.00E+00 4.00E-03 8.00E-03
[ONPG] in cuvette
Ab
sorb
ance
ResultsResults
23 Jan 2007
0
0.2
0.4
0.6
0.8
1
0.00E+00 2.00E-03 4.00E-03 6.00E-03 8.00E-03 1.00E-02
[ONPG]
Absorbance
ResultsResults
Enzyme Inhibition - Non-competitive
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0 0.5 1 1.5
Volume of ONPG added (cm 3)
Ab
sorb
ance