Question

An enzyme-catalyzed reaction was carried out with the substrate concentration initially three hundred times greater than the K_m for that substrate. After five minutes, 1% of the substrate had been converted to product, and the amount of product formed in the reaction mixture was 15 micro moles. If, in a seperate experiment, four times less enzyme and twice as much substrate had been combined, how long would it take for the same amount (15 micro moles) of product to be formed? While answering this question, explain:

a) what is the importance of substrate concentration being 300 times greater than K_m ​?

b) What is importance of only 1% of the substrate being converted in the course of the reaction?

c) Will your answer for the problem be different if the initial substrate concentration is 0.8 of K_m and 40% of it is converted to the product in the cours of the reaction? (Explain, "yes" or "no" is not an answer.)

Answer 1

Even after 5 minutes, you still have a concentration of substrate much greater than Km.

With 4 times less enzyme working at Vmax, it will take 4 times longer to produce the same amount of product, so 20 minutes. The amount of substrate is irrelevant because you have a concentration much much higher than Km.

(a) In order to determine the amount of an enzyme present in a sample of tissue, it is obviously essential to ensure that the limiting factor is the activity of the enzyme itself, and not the amount of substrate available. This means that the concentration of substrate must be high enough to ensure that the enzyme is acting at Vmax. In practice, it is usual to use a concentration of substrate about 10 - 20-fold higher than the Km in order to determine the activity of an enzyme in a sample.