Hi Gunce Goc,

I would like to place my point of view here regarding the results which you have garnered according to the experiment.

As RZ value directly expresses the purity number/purity of protein (by including ratio of two absorbance readings) with respect to its prosthetic group, but not of an enzyme in terms of its activity. In the early research works, it is reported that divalent metal ions like Calcium ions (Ca2+) maintain the protein structure in the heme environment along with the spin state of heme iron that ultimately promotes the optimum conditions for appropriate enzymatic activity. So, If your RZ value is decreasing, then we can speculate that the cofactor might be having ease in terms of its regulatory effect upon the enzyme activity as comparable to the wild type. Variant enzyme activity also gets increased due to the amino acid substitution or change in the aminoacid positions, not in all cases but if specific amino acid positions are targeted in respective to their way of action, like in recombination.

{There are few things which are not mentioned in the question like how much RZ value of variant enzyme got decreased in comparison to wild type and what kind of activity parameters did you specify in accordance to the standardisation of your recombinant enzyme activity through which you seek such enhanced results}

I hope this will help you to clear some of your doubts.

Thank you,

Regards

Hi Gunce Goc,

I would like to place my point of view here regarding the results which you have garnered according to the experiment.

As RZ value directly expresses the purity number/purity of protein (by including ratio of two absorbance readings) with respect to its prosthetic group, but not of an enzyme in terms of its activity. In the early research works, it is reported that divalent metal ions like Calcium ions (Ca2+) maintain the protein structure in the heme environment along with the spin state of heme iron that ultimately promotes the optimum conditions for appropriate enzymatic activity. So, If your RZ value is decreasing, then we can speculate that the cofactor might be having ease in terms of its regulatory effect upon the enzyme activity as comparable to the wild type. Variant enzyme activity also gets increased due to the amino acid substitution or change in the aminoacid positions, not in all cases but if specific amino acid positions are targeted in respective to their way of action, like in recombination.

{There are few things which are not mentioned in the question like how much RZ value of variant enzyme got decreased in comparison to wild type and what kind of activity parameters did you specify in accordance to the standardisation of your recombinant enzyme activity through which you seek such enhanced results}

I hope this will help you to clear some of your doubts.

Thank you,

Regards

Dear Gaurav Sharma ,

First of all, variants of wild type are mutants as you mentioned and size of the replaced residues, of course, are the primary factor for the decrease or increase in the activity. But the Rz values of these variants were so low that we were not even able to measure them.

Based on your reply, it is much more clear to me since secondary activity of the enzyme does not in a need of cofactor - that explains the increase in the secondary activity for these variants.

Thank you so much for the clarification.

Regards,

G

If you what the mutants are and where these are located in the catalase structure, you could get some idea about loss of heme levels in some mutants as well as increased activities.