I am planning to make the wild type enzyme pH tolerant (acidic) without affecting the activity of the native enzyme. Here i am thinking to introduce some residues like lysine or ariginine in place of tyrosine (which is conserved in other protein) the vicinity of two catalytic glutamate residues. Am I thinking in a right way ? please suggest.
Unless you have some really good, accurate models of your enzyme, the only way to know for certain is to actually do the experiment and compare Vmax, Km, etc., of both the modified and unmodified enzyme. The closer to the active site, the more likely it is that mutations will have some effect.
Thank you Greg for your kind reply, I have examined the crystal structure and based on that came to a conclusion that., One of the residue (Tyrosine) in the vicinity of catalytic glutamate seems a potential candidate for the mutation. My query is in general people dont want to disturb the conserved residues, but I suspect if it is mutated to some basic residue like Lysine or Arginine it would be a acidic pH tolerant enzyme. As you said I should perform the experiment, which I agree. Can you enlighten me whether it is always a rule of thumb to not to disturb the conserve residue in general, as here I am anticipating a good effect by this mutation on the basis of crystal structure of the enzyme.
My suggestion is that you compare the active site residues in your protein with those that are found in the same enzyme from an acid-tolerant organism, if such information is available.
You may be correct logically but need to consider the following also -
after changing the residue are you sure regarding the structure of protein, as it may influence ligand binding
The amino acids which are not involving directly in the action they may have the role in directing the ligand to wards active site through different forces.
I can only answer your question by turning it around and stating that the closer an AA is to the active site, the more likely it is that altering it will have some effect on the activity. There are so many exceptions that I would not take that as a strict rule, but just a generalization.
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