I need to perform the recombinant expression of a metalloenzyme. A 6xHis tag was planed to be fused at the C-terminus of the DNA for the protein purification. However, it might be very close to the catalyzing site of the enzyme according to the sequence. I would like to get some software(s) or online tool(s) to predict if the tag would impair the enzyme activity of the expression product. Do you have any advice on the software/tool(s) I can use?
No software will reliably predict how the presence of the tag will affect the activity of your enzyme, since each protein is a special case. I would advise you to design your clone to express a polypeptide with a processing site, such as a Tev protease cleavage site, between the HIs tag and the sequence of the enzyme. Then you can purify the protein using the His tag, remove the tag and compare the activity of the tagged and de-tagged enzyme.
You do not say, what your plans are with the enzyme. If you want to characterize its activity or plan experiment for the turnover of different substrates, then I would recommend to forget about using tags and purify the unadulterated protein.
Anything and everything will influence enzyme activities. I have never worked in a field, in which it is so easy to make a systematic mistake.
In addition, if your enzyme would be used for a pharmaceutical, chemical, or any other purpose requiring large-scale production, it is extremely unlikely that a tag would be used in its manufacturing process. So, if you use a tagged enzyme, the data you collect may show quite different results than the protein purified at large scale.
Another point: you say that you are purifying a metalloenzyme. If you have a his-tag, which is optimized to bind metal ions, floating around near the active center, where the metal ion will be bound, you are very likely to have an interference in your activity.
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