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Unformatted text preview: 7.05 Problem Set 2 1. From Question 3, Exam 1, 2007 2. From Question 1, Exam 2, 2005 3. From Question 2, Exam 2, 2004 4. From Question 5, Exam 1, 2007 5. You have identified a novel protease that cleaves on the carboxyl side of both hydrophobic (i.e., phenylalanine) and basic (i.e., arginine) residues. A. You are interested by its unique specificity. Predict, based upon your knowledge of protease specificity pockets, a residue (or residues) that might be within your novel protease’s specificity pocket (assume that like other proteases, your protease has only one specificity pocket). And account for the unique specificity of your protease. How does its specificity pocket accommodate both hydrophobic and basic residues? B. You suspect that your protease is a serine protease, and you are determined to solve its crystal structure. A post‐doc in your lab is quick to inform you that it would be wise to consider crystallizing your protease with inhibitors. Why is it wise to crystallize your protein with an inhibitor(s)? What are the requirements of the inhibitor(s) with which you will crystallize your protein? C. You successfully solved the crystal structure of your protease. You find that it was indeed a serine protease, and you are now eager to biochemically characterize it. You purify the enzyme and assay its activity by utilizing a substrate molecule that generates a colored product when cleaved by your protease. Your data is summarized in the table below. Construct a Lineweaver‐Burk plot and calculate Vmax and Km. [S] (M) 2.0 x 10‐6 5.0 x 10‐6 6.0 x 10‐5 1.5 x 10‐4 Velocity (μmol/min) 50 100 200 238.1 D. Under what conditions would you expect the velocity to be independent of substrate concentration? E. You find that addition of 100 nM of a compound known as “X” alters the rate of product formation. Values for the velocities of the reaction at the same concentrations of substrate are given below. Calculate the Vmax and Km in the presence of “X”. Is “X” a competitive or a non‐competitive inhibitor? [S] (M) 2.0 x 10‐6 5.0 x 10‐6 6.0 x 10‐5 1.5 x 10‐4 Velocity (μmol/min) (in the presence of “X”) 9.60 22.73 136.4 187.5 ...
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This note was uploaded on 02/06/2009 for the course 7 7.05 taught by Professor Unknown during the Spring '09 term at MIT.
- Spring '09