1.) Aspartate side-chains The role of the aspartate side chains is very interesting to
me. You mention that there is a lack of specificity which means that either
aspartate can take either role. However, I wonder if there are any labeling
experiments that have been performed to see if one aspartate side-chain performs
one role more than the other?
Additionally, if the side chains are mutated, the enzyme won't function, but what
happens if there are more than two aspartate side chains in the active site? Would
there be competition, or would the enzyme still function?
Yes, labeling experiments have been performed to see which aspartate residue
performs which role. Coates et al. studied the neutron diffraction of a hydoxyethylene
based inhibitor bound to the asp protease endothiapepsin on samples soaked in D2O
to exchange hydronium with deuterium. For the transition state complex studied, the
clear result was that Asp 32 was unprotonated while Asp 215 appeared to be
protonated. This suggests that Asp 215 acts as a general base to remove one proton
from the water molecule while Asp 32 donates a proton to the carbonyl oxygen atoms
of the scissile bond.
I’m not aware of any studies that included more than two aspartate side chains in the
active site, but there are studies that show that the orientation of the substrate
molecule in the active site is crucial, suggesting that the new aspartate residue
wouldn’t compete for the substrate but may interfere with the mechanism maliciously
in other ways, for example, by taking up too much space in the active site.
For a great review see, Dunn, B.M., Chem Rev. 2002, 102, 4431-4458.
2.) Hydrophoic residues Is proline the only residue that aspartyl proteases wont
cleave after? Are there other hydrophobic residues that aspartyl proteases will
cleave other than the ones you mentioned? Lysine is so different from the other
'typical' residues that aspartyl protease cleaves after, any idea why this is
included?
I don’t have a specific answer to your question, but the amino acids/sequences that
aspartate proteases cleave are highly dependent on the particular aspartate protease. This
specificity depends on the particular function of the enzyme and it’s elucidate helps
identify its implication in disease. See, for example, the study of the substrate
specificities of the HIV proteases (Poorman et al, Journal of Biological Chemistry, 1991,
226(22), 14554-14561. a table from which I have posted below, which shows some
substrate specificities of HIV-1 Protease.
3.) Most Efficient Environment In normal larger proteins, hydrophobic amino acids
often reside in the center of the protein. Does this mean that aspartyl proteases
need to function in an environment that is either very acidic, or that proteins
haven't been folded yet? Can they hydrolyze larger proteins in their native state?
Yes and no. Some Aspartic proteases, such as pepsin, function in the highly acidic
environment of the stomach where most proteins are denatured. It’s my sense that other
aspartic proteases cleave particular accessible sites on other substrate molecules. In this
sense, this family of enzymes can hydrolyze proteins in their native state.
4.) Localization Do all these enzymes have an active site similar to chymotripsin's
with an oxyanion hole, or some similar apparatus?
Good question. Neutron and atomic resolution work strongly suggest that Asp 32 is
negatively charged in the transition state complex of aspartic protease catalysis. The
negatively charged carboxyl group forms hydrogen bonds with the hydroxyls of a neutral
gem-diol intermediate (see figure below) in a symmetric manner. In contrast to serine
proteases where the enzyme provides the oxyanion binding hole, here the oxyanion
appears that it is part of the enzyme itself, rather than part of the transition state
intermediate. (Paraphrased from Coates et al. Eur Biophys J, 2006, 35:559–566. )
5.) HIV I Protease Evolution All the enzymes mentioned in the aspartyl protease
family are found in eurkaryotes except HIV I protease, which is a viral protein.
Do you know the evolutionary origins of this enzyme? Did it somehow arise from
a human enzyme?
6.) Hydrophobic Specificity Why does this enzyme prefer hydrophobic side-chains?
Is there a hydrophobic pocket that the entire side-chain fits into better? Or is it
something more about how the substrate is led into the active site?
I’ve not been able to find a specific answer for your question but the answer depends on
the particular protease. I would imagine that one of your two hypotheses is correct and in
part depends on the particular protease. Crystal structures exist for many of the aspartic
proteases and I would encourage you to look at them.