PRACTICAL 8: INTRODUCTION TO ENZYME INHIBITION

Using the same enzyme and substrate from last week, trypsin and BAPNA, the effects of an inhibitor on the rate of the reaction were investigated. The rate of the reaction was measured again as absorbance per minute using a spectrophotometer, the concentration of substrate was increased in the different solutions.
The following table of results was obtained:

From this table it can be observed that the absorbance per minute increases for both solutions with and without inhibitor. However this is not the case for solutions D and H, this could be either a mistake when doing the calculations to obtain the absorbance per minute, or maybe the group realizing these solutions placed the cuvettes to quickly in the spectrophotometer and the reaction had not started, resulting thus in a lower value.
The graph obtained is the following:

The pink curve represents the absorption per minute of the enzyme-substrate solution without inhibitor, and the blue one represents the solution with the inhibitor. As it was mentioned before, both curves should increase continuously, however some of the results do not correspond with what was expected, but the following conclusions can be attained by observing the graph.
The absorption per minute is affected by the presence of the inhibitor, this means that less enzyme-substrate complex are found because some of the active sites of the enzymes are occupied by the inhibitor. However, this kind of inhibition, which is called competitive inhibition, can be overcome by increasing the concentration of substrate, and by doing so it would not affect eventually the maximum velocity. This is due to the fact that the enzyme-inhibitor complex is a reversible reaction and as more substrate is added the enzyme manages to overcome the effects of the inhibitor and bind to the substrate. Therefore, if substrate is increased competitive inhibition does not affect the maximum velocity of the reaction.
The case would be completely different in the case of a non competitive inhibition, in this case the inhibitor would not bind to the active site of the enzyme but to a different site called an allosteric site changing thus the structure of the enzyme, and making it unable to bind to the substrate. This situation would affect the maximum velocity of the reaction and it could not be changed by adding more substrate.