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