2. I purify an enzyme, Tea from Starbucksocytes treated with no drug (Control),

Question

2. I purify an enzyme, Tea from Starbucksocytes treated with no drug (Control), Frappe, or a combination of Frappe+NoEmojis. In a separate, but related, experiment, I also test the elution profile of Tea using total cell lysate on an affinity column that uses Frappe on the stationary phase matrix I then repeat this set of experiments, but using the drug Chai, instead of Frappe. My data from all experiments is shown NOTE1: A experiments using Frappe, B-experiments using Chai NOTE2: NoEmoji is a drug that blocks transcription in cells 1lv.f Control 1lv.t Control 0_+Chai _ +Frappe _ +Frappe-NoEmojis -+Chai +NoEmojis 1/[S] 1/[S] Activity _ Total protein Activity Total protein Fractions Fractions a) What is the BEST COMPLETE explanation for the effect of Frappe on Tea? Your answer should include explanations of the effect of Frappe on the enzyme kinetic parameters of Tea, an explanation for the elution profile, and a biochemical explanation for howFrappe affects Tea.

Explanation / Answer

a) From the Lineweaver -Burk Plot, it is observed that in the presence of Frappe, Km of the reaction is altered and their is no change in the Vmax of the reaction this suggests that Frappe is a competitive inhibitor for the enzyme Tea and it binds to the active site of the enzyme and prevents the binding of its natural substrate. Frappe also binds tightly to the enzyme Tea as evident from the high specific activity obtained in one of the eluted fraction as all the other proteins are eluted first followed by Tea in later fraction.

b) From the Lineweaver Burk plot, it is observed that in the presence of Chai as the Km is same as compared to the control and their is a decrease in the Vmax of the reaction. this points out that Chai is a non competitive inhibitor of the enzyme with or without the substrate away from the active site. It changes the conformation of the enzyme rendering it unable for its natural substrate to bind. the elution profile suggests that Chai prevents the binding of Frappe to the enzyme Tea, as in the presence of Chai the enzyme coelutes with the total proteins and is not bound to the column harbouring immobilised Frappe.

c) Since Chai treatment leads to coelution of Tea with majority of the cellular proteins, hence purified Tea from Chai-treated cells will have reduced specific activity as compared to controls. this reduced specific activity is observed due to high amount of proteins present in purified Tea in Chai treated conditions.

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