a) Consider the enzymes in the table below and answer the following questions. A

Question

a) Consider the enzymes in the table below and answer the following questions. Answers should be brief (1-3 sentences) (i) Which enzyme has the highest affinity for substrate? How do you know? ii) Which enzyme converts the most substrate to product in a given period of time? How do you know? Iii) Which enzyme has the highest catalytic efficiency? How do you know? b) You want to load 15 mu g of protein in 10 mu L into one of the 12% polyacrylamide gel well. The protein needs to be in 1 X buffer and in a total volume of 0.100 ml. You are given a 5 mg/ml protein solution, a 20X sample buffer, and distilled water. How much of each would you mix together to make required volume?

Explanation / Answer

6a. (i). A large value of Michaelis constant, Km, indicates lower substrate affinity of enzyme and vice-versa.

Here, enzyme C has the lowest Km value (4.5 x 10-6 M), hence enzyme C has the highest affinity towards substrate.

(iii). Catalytic efficiency of an enzyme is given by the ratio of its Kcat to its Km i.e. Kcat / Km. For the given enzymes, catalytic efficiency is given as:

Enzyme A: Kcat / Km = 1.4 x 104 / 9.5 x 10-5 =   1.47 x 108

Enzyme B: Kcat / Km = 1.0 x 107/ 3.5 x 10-2 =   2.85 x108

Enzyme C: Kcat / Km = 7.5 x 102/ 4.5 x 10-6 =   1.66 x108

Hence, enzyme B has the highest catalytic effciency.

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