Proteases

In this step we will analyze thermolysin, a zinc protease. I suggest you first read the nice text put together by Jolanta Grembecka. In case this page is unreachable, you can use the local copy. (The pages contain several typos, but you are smart enough to not be disturb by that).
After reading these pages, answer the following questions:

Question 27:

• Why is zinc a good atom for active sites?
• Which zinc binding enzyme is used to produce aspartame? (what is aspartame? and how can one produce a peptide with a protease?)
• Why are there two mechanisms for the action of thermolysin? And which experiments could solve this problem? (Think about partial charges needed to perform the action).
• Look at the NOTE below on protease sub-site nomenclature.
• What can you find about the specificity of thermolysin?
• Which residues form the active site? Which residues are important?

Figure 38. Get the file 8tln.pdb from the PDB. Answer the questions listed at the bottom of the page.

Thermolysin is the molecule that is used to make aspartame. Lets do some serious structure - function relation thinking. Use these questions as a guide, but be prepared for much more difficult questions from the assistant...

• Look up the specificity of thermolysin (i.e. what are the sequence rules for a good substrate).
• Which dipeptide is bound in the pocket of the 8tln file for thermolysin?
• Look at the possible thermolysin mechanisms. Where roughly (in space) does the cleaving take place?
• So, roughly where do you expect (in space) to find the P1 and P1' residues?
• And where do you observe the two amino acids of the dipeptide?
• Why can this dipeptide not occupy the S1 and S1' pockets?
• What is aspartame?
• How is aspartame produced?
• What part of aspartame goes to which sub-site?
• Does this agree with the thermolysin substrate specificity rules?
• How can enzymes be improved in general?
• What kind of mutations could make neutral proteases produce aspartame quicker?

Question 28:

• Which sub-sites are 'filled' by the dipeptide in 8tln?
• Does this agree with what you found out about the specificity of thermolysin?
• Which residues make up the S1' sub-site?
• Which residues make up the S2' sub-site?
• Describe the thermolysin domain structure.
• In order to cut a peptide, the two domains of thermolysin must move in a hinge like motion. Find the hinge.