5 amino acid residues at the catalytic/active site were found to be interacting with the substrate. How to predict the effect of mutations on activity and stability? Also, Is it logical to opt for site saturation of each residue individually in order to screen for the best mutant?
Hi Rajul, the best way to analyze your enzyme mutants is to do some kinetic studies on them.
Hello Steingrimur Stefansson and Kou Hayakawa. Thank you for your valuable replies. Actually my query was about prediction of the effect of mutations on my enzyme (any in silico approaches).
Hi Rajul, I'm certain that the in silico approach will get you some great data.
But it always comes down to making the enzymes and testing them in wet lab conditions to prove your in silico predictions.
Be aware that nature has a way of kicking you in the backside when you think you have thought of everything.
Hi Steingrimur, I agree with you on that. We want a prediction in order to have an idea what kind of mutations we should go for. I have 5 residues as target to mutate so, should each be replaced by rest 19 a.a's or what actually will be helpful ?
The ultimate goal is to take the mutants to wet lab (i.e to purify and characterise), but not all the mutants (some may be of no use).
And I guess in silico approach will help somehow.
Apparently, I have shortage of time and yes, I am very much aware of the nature's habit, and that is why seeking help from experts (like you) to actually streamline my work.
Thank you and have a great day!
Rajul,
To the in silico problem you probably need to take care in comparing the functional motifs of both mutant and non mutant enzymes by analyzing their respective sequences in computational tools as PROSITE, BLOCKS, ELM...
To the confirmation through in vitro tests (in wet lab) you can try to compare the enzyme kinetics. After to estimate the enzyme activity in relation to different substrate concentrations, you can plot both Michaelis-Menten and Lineweaver-Burk graphs, analyze the obtained constants, and evaluate possible difference in terms of enzymatic performance.
Thank you Wendell,
I am trying to mutate 5 a.a residues which were found to be interacting with substrate (as depicted from crystal studies). What approach should I use- whether to mutate all or mutate individually (site-directed, multi-site directed or saturated mutagenesis?
Hi Rajul, do you have the resources to generate and isolate at least 1 of the mutant enzymes and test its activity?
If you can and it behaves in vitro as predicted in silico, then you can have the leverage to ask for funding to generate more mutants.
If you can't do that, then I am afraid that all your hard work will just be stored in a computer somewhere.
But, there's a possible way out of this situation. Use BLAST and Swiss-Pro to study all the similar enzymes across species and sequences of their active sites.
If you find a natural mutation that corresponds to your in silico predictions, and if its catalytic parameters are known, then you don't need to do anything. Your studies are vindicated and you don't have to make any mutants de novo.
Hello Steingrimur,
Thank you so much for your valuable inputs, it means a lot!
I have created and purified many mutants. After characterization, simulation studies support the in-vitro data. As of now, I am using GROMACS for m designing and engineering the selected clones.
Rajul-- To predict enzyme activity you need to predict three K's: Kcat, Km and Keq.
Kcat is proportional to the binding affinity of the enzyme to the transition state of the reaction. You can calculate it by modeling the t-state onto the structure and looking at what your muattion will do to the stability of that complex. We have to assume that nothing else moves.
Km is the ligand binding constant, so you would get the change in Km in a similar way. Model the bound complex and ask what your mutation will do to it, if anything.
Keq is the stability of the enzyme. The enzyme function depends on how much of it is folded, and this will depend on distal mutations if the stability is marginal. Otherwise, if the enzyme is robust, only very detrimental mutations will have an effect.
This is a simple and non-quantitative answer. To get more real you may have to do some more serious computation, such as MD simulations. But I would keep it simple and non-quantitative. You will be able to tell if a mutation will have a negative effect, and maybe even a positive effect, but to get a quantitative answer using in silico methods is still beyond our abilities.
Thank you Christopher.
I was looking up to qualitative levels only and your suggestions are actually what I was seeking for. I am interested in fishing out the detrimental mutations for my enzyme (in silico way) and ultimately doing the quantification (wet lab experiments).
The "affinity maturation" tool in Maestro Shrodinger can be retrofitted to quickly do in silico mutagenesis screening of the effects of mutations on affinity (for substrate) and stability. It is a paid-off software though. I would recommend doing a 2-3X saturation approach of the residues in the active site. 4X and greater leads to explosive numbers unless you use a reduced alphabet.
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