KE0026 Biochemistry Labs

Computer lab: Enzyme structure and function

Goals: to understand the structure and function of trypsin.

In this lab you will use SWISS PDB VIEWER (SPV) to investigate the structure and function of an enzyme. Your lab report should look a lot like the ones for the wet labs, but make sure that you include in it the answers to the questions below.

Start by downloading a pdb file for trypsin (1TPO from the Protein Data Bank).

Use SPV to load the pdb file and look at the structure. (Remember, there is a webpage with the manual on how to use SPV, so use it!)

What is the reaction catalysed by trypsin? What does the substrate look like? Are some substrates better than other substrates?
Look at the structure of trypsin (hide the side chains and the main chain oxygens). Guess where the active site is, zoom in on it and look at the residues there. If you can not find it by guessing, look in the book (remember chymostrypsin and trypsin are very similar).
Identify the catalytic triad. Describe the interactions between these residues, stating the distances between the atoms involved. A picture would help here. (Hint: first decide what the word "interaction" actually means.)
Now download the file for 2PTC from the PDB and load it into SPV. This is the structure of the trypsin-bovine pancreatic trypsin inhibitor (BPTI) complex. What parts of the BPTI polypeptide chain interact with trypsin? (Hint: coloring trypsin and BPTI differently will help you see this. Also, you can look at the picture of the complex in the course's Structure Gallery.) Look for specific interactions between trypsin and BPTI: give one example of a hydrogen bond, and one of a van der Waals interaction (give atom/residue names and the distances between these atoms).
Which residue/residues are most important in forming the substrate specificity site of trypsin? What characteristics of these residues are important? (What kind of interactions do these residues make with the inhibitor? Is size/shape important here?).
How does BPTI inhibit trypsin? (Is it competitive, non-competitive or uncompetitive; and why?)
Summarize what you have learned above, and apply it to the reaction mechanism of trypsin. Discuss how trypsin recognises its substrate and how it performs catalysis. You may find it helps to include a picture to support your discussion.

Some tips and clues
a) The SPV manual is there for use, so - use it.

b) When looking at overall structures it is a good idea to turn off the side chains.

c) When looking at the active site it usually helps if you look only at residues that are close to the active site. For example, the program allows you to choose for display only residues within a given radius of an atom. You can use this to make a picture of with just the residues in the active site.

d) zoom in and zoom out and try to look at the structure while rotating it - it increases perception of depth.

If you run into problems, don't hesitate to ask for help.

Lab index

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