Exercise 3.
Analysis of the catalytic site of a an enzyme

1. Load structure 1CDE of enzyme GART (GlycinAmide Ribonucleotide Transformulase)
2. Do a quick check of Ramachandran Plot
3. Do a quick check of the structural motif using ribbon representation - this is a mixed alpha/beta structure
4. Identify the main players of catalysis
   Hide structure
   Display substrate GAR (GAR222) and cofactor tetrahydrofolate (DZF225)
   Add to view residues within 5 Å from the terminal N-atom of GAR222
   Hint: use 9th button of Toolbar, follow instruction in Toolbar
   Repeat this step but instead of "Add to view" use "Add to selection"
   Do you see a His and an Asp?
   Answer: yes, His108 and Asp144; these are catalytic His and Asp
   Add to view and Select residues within 5 Å from the 2 N-atoms of His108 side chain and from the 2 O-atoms of Asp144 side chain
   Did a new His come into the picture? Which one?
   Answer: yes, His137; this is a conserved His
   Keep only the His and Asp residues and GAR222, DZF225
5. Identify the presence of favorable Coulombic interactions between His108-Asp144, His137-Asp144
   Are there unfavorable Coulombic interactions between His108-His137, His108-GAR222?
   Coulombic interactions between charged side chains are present when the charge sites are < 6 Å apart
   You can use Display Label kind "Clear user labels" to get rid of labels
   Answer: Favorable Coulombic interactions, distances between possible charges for His108-Asp144 are 3.64, 4.30, 4.35, 4.36 Å for His137-Asp144 are 3.90, 5.62, 5.91 Å. Unfavorable Coulombic interactions, His108-GAR222, distances between possible charges are 4.12, 5.31 Å for His108-His137 is 5.74 Å
   Will these interactions affect the pKa values of His108, His137, Asp144, and GAR222?
   Answer: definitely
6. Mutate and minimize
   Hide all residues with the exception of Asp144
   Add to view all residues within 5 Å from each of the 2 O-atoms of the carboxy group
   In Tool Bar click on the 12th button (mutate button)
   Click on Asp144
   Mutate Asp144 to Glu using the Pop up Window
   Check if there are van der Waals clashes
   Answer: of course, Glu is longer than Asp and the catalytic site is packed
   Make sure all residues are un-selected and select only Glu144
   In Tool Bar Tools Energy minimization
   Visualy inspect the result, then check if the clashe is gone, check if new clashes have been created
   Answer: indeed new clashes showed up
   In Tool Bar Tools Compute H-bonds - check if new H-bonds have been created?
   Answer: no
   You can repeat the minimization by including 2 residues, e.g. Glu144 and His108, but keep in mind that if you include more residues the local structure will change - after all an Asp to Glu mutation in the catalytic site may not be a good idea!!!