Project 4 – Exploring Enzymology in the BRENDA database

Instructions

In this exercise, you will simply have to use a database called BRENDA to look up some of the specific kinetic properties of your unknown protein. On the BRENDA page, numbers with the format: #5# indicate that the data refers to the enzyme isolated from organism 5. The list of organisms at the for any given enzyme is found at the top of the BRENDA entry for the protein. Numbers with the format <5> indicate that the data derives from reference 5. References are found at the end of the entry.

To get to the BRENDA database:

• Go to the ExPASy web page (http://www.expasy.ch)
• Click on the link to the ENZYME program
• Under related tools and databases, click on BRENDA
• Enter the name of your enzyme in the search window and press query
• One or more E.C. listings should appear. To enter the database, click on the E.C. number of the correct enzyme in the query table.

You can also access this page from the BRENDA link on the Nice-Zyme page within ExPASy.

Information to find in the BRENDA database entry.

a) Use the biochemical literature or biochemistry textbooks to find the mechanism that has been proposed for your enzyme. If you take the mechanism from a source – be sure to include an appropriate reference.

b) What are the ranges of specific activity that have been measured for this enzyme?

c) Are there different specific activities reported for the enzyme from the same organism? If so, why might the specific activity vary?

d) Why do you expect the specific activity of the same enzyme isolated from different organisms to be different?

e) Go to the section on KM. Pick a substrate that has been used in several studies. What are the ranges of KM’s that have been measured for that substrate? Explain what leads to variation in the kinetic parameters measured between these different studies.

f) What is the pH optimum of your enzyme? You have already explored the structure of your enzyme and the overall mechanism (part a above). Relate the pH optimum to some aspect of the enzyme mechanism.

g) Identify two competitive inhibitors for the enzyme from the table of inhibitors. Remember that competitive inhibitors usually look like the natural substrate.