Hi Subbu,
Here's my two cents.
Your project sounds really interesting.
You mentioned in your email that the active site and associated waters
in the active site of the wild type and mutant enzymes are
"identical". It's worth noting that even very small (on the 1/10th of
an angstrom level) structural perturbations can have impacts on
protein function. When high pressure is applied to some enzymes, their
activities can be affected by factors of 2 or 4, or perhaps even more.
The structural deformations due to high pressures (1000 x atmospheric)
are often on the sub-angstrom level.
If you have sufficient crystals, I might be tempted to re-solve the
mutant and wild type structure a few times with fresh data, and get a
feel for how much error exists is in the position of key residues.
This might tell you how close the geometry of the two active sites
really are.
Good luck! and all the best,
--Buz
On Jun 11, 2008, at 8:21 PM, Narayanan Ramasubbu wrote:
> Dear all:
> I have a single residue mutant whose enzyme activity is about 50% of
> the wild type. Interestingly, the mutation
> is in a region that involves a secondary site but not the active
> site. The two structures with or without ligands
> fit well (0.18 A) and the metal binding and cofactor binding sites
> are all preserved in the mutant. The one difference
> noticed is that the ligand does not fill the active site (partially
> occupied subsites) unlike the wild type where all the
> subsites are occupied. Water structure around the actives site
> residues are "identical".
>
> I looked at the electrostatics and both surfaces look similar (not
> an expert).
>
> There are some residues whose sides chains show some positional
> disorder and these residues are at the edges of the
> active site.
>
> The resolution of the both data sets are 1.5A.
> The mutant enzyme was derived by MR.
>
> One another possibility that I want to look at is to compare the
> compactness of the two enzyme structures.
> What is the best way to compare that? I am wondering whether the
> "breathing" that was mentioned for some enzymes
> might be playing a role in the mutant enzyme.
>
> Also, I would appreciate comments on other possible explanations for
> this unusual (?) behavior.
>
>
> Thanks a lot
>
> Subbu
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