Q4. When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme o-diphenol oxidase catalyzes the oxidation of phenols in the apple to dark-colored products. To determine the Vmax and Km for this enzyme you run several reactions under three different conditions:
When the enzyme acts alone on its substrate catechol converting it into o-quinone, which is then further oxidized to dark products. (A)
When the enzyme acts in the presence of an inhibitor para-hydroxybenzoic acid (PHBA), which binds the same site as catechol but is not acted upon. (B)
When it acts in the presence of an inhibitor phenylthiocarbamide, which binds to a copper atom in the enzyme which is essential for its activity. (C)
a. What is the Km and Vmax for this enzyme with its natural substrate?
b. What kind of an inhibitor is PHBA and phenylthiocarbamide? Why?
c. Assuming that the enzyme present in the system had a concentration of 10-6 M, calculate its turnover number.
d. Does PHBA combine with E, with ES, or with both? Explain
OH 1/2 02 + H20 OH catechol O-quinone H,NS OH (C) » HOOC NH para-hydroybenzoic acid PHBAI phernylthiocarbamide From all your-experimental data you construct the following plot: 1/v phenylthiourea th PHBA 50 No inhibitor 20 ISI (1/mM)
