We have isolated β -Glucuronidase (cleaves β-glycosidic bond) enzyme from E. coli and two other species. These enzymes were then tested for their glucuronidase activity on Glycyrrhizin (structure below) as substrate. It has been found that β -Glucuronidase obtained from E. coli is showing good enzymatic activity and others are not.
Now, we want to prove the same study using in-silico tools. Is it possible to model the enzyme substrate reaction via computational methods. If yes what are the tools required for the same? Can we do it on freeware tools?
Please suggest me some way to sort out the problem.
Docking of substrate to enzymes can be performed using Glide in Maestro or using Autodock software. You should have an idea of where the binding site is, otherwise it will take you some time to locate it computationally. Scoring values you get from docking can give you an estimation of relative binding of substrate to each enzyme.
There exist also other methods such as molecular dynamics simulations which are more accurate in terms of the information you get to analyse, but a little more expensive computationally, especially in finding force field parameters for substrate and I am assuming you already have the 3D structures for your molecules, otherwise this would be another difficulty.
AMBER is a better method for this molecules, the MM method is the first option in this case,
Yes you can... but of course it would be impractical to use full QM method if you have limited computational resources... and even if you have a sufficient resources, one practical approach would be to use ONIOM method...
http://pubs.acs.org/doi/abs/10.1021/ct050289g?prevSearch=%2528ONIOM%2529%2Band%2B%255BContrib%253A%2BMorokuma%252CKeiji%255D&searchHistoryKey=
Dear Sinisa,
Thank you very much for your valuable time and such a nice response
Dear Omprakash Tanwar,
The first and most important question is how did you measure the enzyme activity towards glycyrrhizin?
HI, I have made a dock of glycyrrhizine to E.coli GUS (pdb code 3LPG). It seems your "substrate" cannot enter into enzyme substrate binding pocket for it is too big to. I have some ideas how to make a good paper of that findings. Please write me if you are interestd in )
Dear Sir,
Thank you very much for interest in my query? I did the same study with E. coli beta-glucuronidase (PDB id 3K4A) and found the encountered the same problem.
In our study we isolated the alpha-glucuronidase enzyme from three species; E. coli, P. chrysogenum and rhizopus oryzae. The enzyme was then purified and lyophylised. The enzyme activity was checked with the glycyrrhizine. One of the enzyme had a great Km value than others. Now we want to prove the same pattern of activity with the help of in-silico tools by modeling the alpha-glucuronidase on the substrate glycyrrhizine.
Please help us regarding the above mentioned problem.
Regards
Dear Omprakash Tanwar,
I was asking you about the method of he enzyme activity measuring for if you measure directly the amount of glucuronide residues cleaved then you can talk about substrate specificity of your enzymes. On the other hand, if you use one of the indirect methods like the popular method based on the developing yelllow color of p-nitrophenol released from the substrate, p-nitrophenyl-beta-D-glucuronide then your glycyrrhizine migh simply inhibit the reaction with a certain type of the enzyme.
I wish you every succces in your work,
regards,
N.S..
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