For information, I'm work with an enzyme from bacteria. Unfortunately, almost all previous reports only discuss about the active site of this enzyme in yeast. I already tried to search information and do Blast to find out the template and extract the information. I also have done catalytic prediction by using software and compare this with template information.
But, I have a bit doubt about my result and how to make sure my analysis is correct? or is there another way to determine active site of enzyme. Thank you
Hi there,
Have you run a blast with the yeast protein to find homologs in the different kingdom? if yes, do you get your bacterial protein? If so you should spot the very conserved amino acids in all homologs among which you would find the catalytic ones. Is the structure of the yeast enzyme known? If yes, you could map the conserved residue on the structure and define the active site. You can also try to generate a structural alignment with your bacterial protein or generate a structural model of your bacterial protein...
you can study the nature of active site easily by a simple experiment. I will try to explain: incubate the enzyme with different metals separately. then add to the substrate . metal giving you best activity is the enzyme co-factor. then try EDTA and chelating agents, if inhibited enzyme activity so the active site is metallo-type. if metals not gave these results then study the thiol active agents and so on.....
Since you have already performed the catalytic prediction, you can now move to the wet lab. Generate deletions with and without the predicted catalytic domain and check for maintenance of activity when your predicted domain is present. Depending on the length of your protein, this can be an easy task.
Perform directional cloning. Prepare the plasmid you will use with two different enzymes. Use forward primers for the specific region of your gene with the additional restriction site for the 5' end and a reverse primer with the restriction site for the 3'end.
Check that you have not introduced any stop codons, ligate transform, assay for activity.
Alternatively, you can have the specific regions synthesized and inserted into the vector of choice, transform bacteria and test for activity.
There are several controls that you must keep in mind should you decide to go in either direction. It all depends whether you believe tht you have exhausted the bioinformatics options.
Good luck
I think Fedora's suggestions with deletions might lead you to the wrong conclusions. Presumably you know what the enzyme's substrate is? If you know any specific inhibitors this is also good. In the latter case you can try X-ray crystallography with and without inhibitor which should be binding in or near the active site. This latter technology has become more reliable (making crystals) and faster in recent years. If you don't have someone with this expertise you should be able to interest someone at another facility to help you in a collaboration.
Essam's suggestions for classifying which class of enzyme you are dealing with (metal-, thio- etc. is also a routine test that should be done and will deliver valuable information, but be cautious that the effects seen are not just general protein denaturation (e.g. when using thio reagents) rather than specific effects at the active sites
Hello
CASTp, Autodock vina, Swissmodel, Qsite, Chimera, RasMol or JMOL, BIOVIA Discovery Studio.
Good Luck.
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