We discovered in Step 4 through a pairwise sequence alignment between R- and S-HPCDH that 1) the residues that bind the hydroxyl moiety of R-HPC and assist in catalyzing the reaction are conserved and 2) that the most dramatic change is that the positively charged residues (two Arginines) that bind the sulfonate moiety of R-HPC are not conserved. Thus, it seems logical to hypothesize that the sulfonate and methyl binding sites have exchanged positions in the enzyme. S-HPC would then be expected to bind to S-HPCDH with the hydride directed toward NAD in contrast to how it binds in R-HPCDH (recall Step 3).
Nevertheless, we are still left wondering which specific positively charged residues are responsible for the basis of stereospecificity. This knowledge is useful to understanding the evolution/re-engineering of proteins and for a deeper knowledge of enzyme catalysis. There are 15 Arginines and 4 Lysines in S-HPCDH. An examination of all the Arginines (Arg) and Lysines (Lys) might be expensive both in money and time. The sequence alignment, though useful in several other contexts, fails to give us an answer as to which residues cause the switch in stereospecificity. Therefore, in Step 5 we constructed a comparative model of S-HPCDH using R-HPCDH as a template. The structural model can in many circumstances help narrow the scope of investigation by identifying positively charged residues that are positioned to properly bind the sulfonate group of S-HPC.
In this final step, we will visualize the structure and identify the residues that determine the basis of stereospecificity.
We then need to give some context to all the positively charged residues by examining how close they are to the active site.Display conserved Tyr148 Display NAD
Tyr148 is displayed in red to better distinguish it from other residues.
Look for 2 positively charged residues that point into the active site and that are relatively close together (imagine them spaced just far enough apart to bind two oxygen atoms of the sulfonate group). One Lysine is intimate with the active site, is part of the catalytic triad and can be discounted as a residue responsible for the basis of stereospecificity.
If you turn off the ribbons (uncheck the box) and create a surface representation of the entire protein except for the positively charged residues, this should help in determining the correct residues.Calculate/Display Surface
The cofactor NAD, represented in stick form, is a large molecule and only gives an idea of where the active site is located if we look at the correct end. In this model, the adenine moiety is more accessible while it appears that the nicotinamide moiety is more buried. Recall that the nicotinamide part is what receives the hydride and therefore must be part of the active site. Some rotation and perhaps zooming in on the complex may be required.
Fill in your answer below (ex. Arg192 Arg196)