Hi, Others have suggested very rational possibilities, so I would like to mention the odd one out:
It may sound strange but I have to ask - 50% activity as measured how? Many biological assays have intrinsic experimental errors around 10%. If you add on top of this the difficulties encountered when trying to properly quantify protein concentration (needed to measure specific activity) then you can have additional 10-20% error. If you also consider the possibility of slightly different expression environments for the native vs mutant - the 2-fold difference may not be significant at all. Alternatively, you can have active enzyme mixed with inactive one (happened to us several times) - and the activity for the competent enzyme form appears to be a fraction of its true value because the 'active' enzyme is diluted n-fold with the 'lazy' enzyme. What often works in this case is an additional purification step based on specific affinity towards substrate or cofactor. That's not to say that your assay results aren't right - merely to suggest a few alternatives (i.e. there is no significant difference, but it appears to be there due to experimental uncertainty or additional factors). How many independent enzyme preps were used to measure this variation? As you can easily see, repeating the assay N times with the same enzyme preparations is a good practice, but it's not sufficient to substantiate relatively small differences such as this one. You can also seek independent biophysical validation - for instance, if you have an option to measure binding of one substrate by e.g. calorimetry then you can determine the value of N experimentally and thus establish how much of your enzyme pool is in a 'competent' state. Cheers, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Narayanan Ramasubbu Sent: Wednesday, June 11, 2008 8:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Activity of a mutant enzyme compared to wild type - puzzle 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
