Question

Your colleague discovered a potential small molecule reversible inhibitor of the enzyme.  How would you set up an experiment to determine the type of inhibition this small molecule has on the enzyme?  

Answer 1

SOLUTION:

In competitive inhibition Enzyme combines with Substrate forming enzyme substrate complex.

E + S ----} ES

If inhibitor is present, the Inhibitor binds to the same site where substrate binds and form enzyme inhibitor complex.

E + I ----} EI

The competitive binding of inhibitor is higher than that of substrate binding.

In our experiments considering the above facts we divide our experiments in different groups.

Group 1: Enzyme + Its natural substrate

Group 2: Enzyme + small molecule inhibitor

Group 3: Enzyme + its natural substrate + small molecule inhibitor

Group 4: Enzyme + a non-inhibitor molecule + Its natural substrate (negative control)

Group 5: Enzyme + a potent inhibitor + its natural substrate (positive control)

We have designed our experiments into 5 different groups with 2 control groups. In positive control we will be incorporating an already proven inhibitor of the enzyme under study. In negative control we use a molecule which does not possesses inhibitory activity on the enzyme. Comparing the results from these five groups and calculating the significance using statistical tool we can reach in a conclusion that whether our small molecule actually have competitive inhibition on the respective enzyme.