Uw Bioen 457 Exam Key

4. You are a molecular bioengineer given the task of engineering an enzyme to work faster.  Your application requires a substrate concentration that is 3 orders of magnitude higher than the substrate encountered by the enzyme in the biological setting where it evolved to work.  What would your strategy be to engineer this enzyme to work most effectively in your application – and why? (10 pts)

The higher substrate concentration in your application allows you to maintain the same percent bound of enzyme-substrate at higher Km values.  By engineering a lower affinity of enzyme-substate interaction, you can decrease the dGactivation, and engineer a faster kcat.  Therefore engineer a higher Km for your enzyme while maintaining transition state affinity.

8.  A company is producing a new enzyme product that hydrolytically degrades the following substrate. They have hypothesized that adding a general base and a general acid would increase the enzyme’s catalytic efficiency.

[pic 1]

a.  Draw the transition state structure for this reaction, and include a potential general base and a general acid that could be used to achieve enzyme rate enhancements. (10pts)

[pic 2]

b. What Michaelis-Menton analysis would you suggest to determine whether your general base was functioning as you designed, i.e. what changes in Michaelis-Menton parameters would you expect? (5pts)

• A general base should result in an increase in Kcat
• No change in Km and Ki

c.  What Michaelis-Menton analysis would you suggest to determine whether your general acid was functioning as you designed, i.e. what changes in Michaelis-Menton parameters would you expect? (5pts)

• A general acid should result in an increase in Kcat and a decrease in Ki
• No change in Km

9.  The following data has been collected in an experiment to determine a protein’s folding free energy.  Determine the folding free energy of the protein by the linear extrapolation method. (10pts)

Fluorescence Intensity at Peak           [GnCl]

1.2        0

1.4        1.0

1.8        2.0

3.7        2.5

5.7        3.5

6.2        5.0

6.3        6.0

[pic 3]

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