• Describe experimental enzyme inhibition and how it leads to a deeper understanding of enzyme mechanisms.

An enzyme is a folded protein. They play a very crucial role in all the life processes. But the function of enzymes is needed only in certain conditions. When the conditions don’t apply, the activity of a particular enzyme can be dangerous. The enzymes are strictly regulated at various levels in all the biological processes. When the enzyme’s effect is not needed, they are inhibited, such molecules which inhibit enzymes function are known as inhibitors. Inhibitors work by binding to enzyme there by slows down or halt the catalysis. So, Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition.
Inhibitors can be REVERSIBLE or IRREVERSIBLE.
How to characterize enzyme inhibition experimentally?
1) Take four different test tubes and add different concentrations of catechol, which is a substrate (here).
2) Now, add a fixed amount of enzyme to tube A and measure the change in absorbance (OD) at 540 nm, at regular intervals for several minutes.
3) Record the average change in OD540 per minute (Δ OD540).
4) Because the OD is directly proportional to the concentration of the products, we can use it as a measure of the rate or velocity of the reaction (Vi)
5) Repeat with the other three tubes.
6) Now repeat the step 1, but this time add fixed amount of a solution of PHBA, ( para-hydroxybenzoic acid) to each of the four tubes.
7) Now, take OD for these tubes.
8) Plot Lineweaver-Burk plot for the obtained OD, (Vi) values for + and – PHBA vs. [S]. (convert V to 1/V and S to 1/S).

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