<p>Your title says <code>holistic</code>. This is a tad problematic as there's layers upon layers. Say, post-translation regulation, inhibiting metabolites, interacting protein etc. That is why when talking of an enzyme inhibitor, at the biochemical level one speaks in terms of k_i (inhibition constant), while at the cellular level (&quot;holistic&quot;) one talks of IC50.</p>
<p>This could be a biocatalysis/synthetic biology problem (make an enzyme better) or translational medicine problem (why is this mutation pathogenic).</p>
<p>In terms of <em>in silico</em> enzyme design, one can have all sorts of issues that need to be addressed:</p>
<ul>
<li>catalysis</li>
<li>side reactions</li>
<li>stability</li>
<li>aggregation</li>
<li>solubility</li>
</ul>
<p>Also there's the expression problems, which may be due to subpar promoter, insufficient cofactors and poor codon usage.</p>
<p>For clinical variant assessment, a variant may:</p>
<ul>
<li>destabilise leaving to degradation</li>
<li>remove a post-translational modification</li>
<li>disrupt the active site, affecting catalysis</li>
<li>lose a binding partner</li>
<li>result in incorrect localisation</li>
<li>result in solubility issues</li>
</ul>
<p>So force-field scores are not the sole solution. Furthermore, a forcefield method gives one a ∆∆G (difference in relative Gibbs free energy), not a K_M (Michaelis constant) and a k_cat (catalytic rate), although it is somewhat possible to calculate these, or a relative variant of these.</p>
<blockquote>
<p>a variant with just 1 or 2 variants</p>
</blockquote>
<p>A variant protein can have one or two <em>mutations</em> (e.g. G10R) in its sequence. A <em>mutant</em> and a <em>variant</em> are near synonyms, but variant is generally intended to be a mutant in a population.</p>
<blockquote>
<p>Does these application be used for such purpose?</p>
</blockquote>
<p>For enzyme design MD simulations are not generally the first step. Rosetta pmut_scan for example is a nice starting step as the mutational landscape of a protein is N^19, while a MD simulation takes weeks and 6+ core per single variant.
This calculates only an increase in stability (relative difference in Gibbs folding energy), not reactivity. For that you can use a transition state, whose results are inaccurate, but majorly affected by the set-up, i.e. junk-in junk-out plays a big part. There's a saying 'a week in the lab saves you an hour at the library': this is backwards for sarcasm as it happens too often, namely that is background research is always a lifesaver. An QM-MD simulation on the wild type reaction definitely can help in elucidating things if it is not clear.</p>
<h2>Online tools</h2>
<p>Generally, however, there are resources out there to do many stability calculations already.</p>
<p><strong>SynBio</strong> If you are interested in picking a residue or two to make an enzyme more stable, without resorting to a thermophilic enzyme, <a href="https://pross.weizmann.ac.il/step/pross-terms/" rel="nofollow noreferrer">PROSS</a> is a very nice tool.</p>
<p><strong>Clinical</strong> (Caveat: If your variant is a variant that affects you, a friend or a relative, you/they should speak to a genetic councillor and not do research on it yourself) There are several tools that can describe a variant at the protein level. Do remember that ∆∆G is a potential so more negative is more stable —except in SDM where the score is inverted.</p>
<ul>
<li><a href="http://www.sbg.bio.ic.ac.uk/%7Emissense3d/" rel="nofollow noreferrer">Missense3D</a></li>
<li><a href="https://venus.sgc.ox.ac.uk" rel="nofollow noreferrer">Venus</a></li>
<li><a href="http://miscast.broadinstitute.org/" rel="nofollow noreferrer">Miscast</a></li>
<li><a href="http://www-cryst.bioc.cam.ac.uk/%7Esdm/sdm.php" rel="nofollow noreferrer">SDM</a></li>
</ul>

CC BY-SA 4.0
2021-04-16T12:09:51.217Z
2021-04-16T13:35:12.523Z
2021-04-16T13:35:12.523Z
Matteo Ferla
<p>I'm currently working on a program which will determine the activity difference between an original enzyme and a variant with just 1 or 2 variants. Of course, I'm not talking about the &quot;kinetic activity&quot;, but some indications of a probable activity difference should be given. The system has to be sensitive enough to determine the probable activity difference between unthought-of variants.</p>
<p>From the information that I've got from my research, there is two option. I can use one of 3 quantitive enzyme reaction analysis tools (such as EVB, FEB and LRA) or their hybrids.</p>
<p>EVB =&gt; Empirical Valence Bond</p>
<p>FEB =&gt; Free Energy Perturbation</p>
<p>LRA =&gt; Linear Response Approximation</p>
<p>Source:
<a href="https://www.researchgate.net/post/Can-the-activity-of-proteins-be-studied-using-molecular-dynamic-simulations" rel="nofollow noreferrer">https://www.researchgate.net/post/Can-the-activity-of-proteins-be-studied-using-molecular-dynamic-simulations</a></p>
<p>Now, my questions are: - Does these application be used for such purpose? - For an accurate prediction of  enzyme activity difference, are these programs good enough or require developement? - Which one of them can give the best result? - And lastly, are QM/MM methods a must for an accurate answer?</p>
<p>I'm a high school student so sorry if my questions sound dumb.
If you can answer my inquiry's I would appreciate it:)</p>
<p>Thanks</p>

<proteins><protein-structure><proteomics><structural-biology>
Holistic enzyme activity determination with computation
# Holistic enzyme activity determination with computation

(Asked by: username_0 on 2021-04-12)

I'm currently working on a program which will determine the activity difference between an original enzyme and a variant with just 1 or 2 variants. Of course, I'm not talking about the "kinetic activity", but some indications of a probable activity difference should be given. The system has to be sensitive enough to determine the probable activity difference between unthought-of variants.

From the information that I've got from my research, there is two option. I can use one of 3 quantitive enzyme reaction analysis tools (such as EVB, FEB and LRA) or their hybrids.

EVB => Empirical Valence Bond

FEB => Free Energy Perturbation

LRA => Linear Response Approximation

Source:
<https://www.researchgate.net/post/Can-the-activity-of-proteins-be-studied-using-molecular-dynamic-simulations>

Now, my questions are: - Does these application be used for such purpose? - For an accurate prediction of enzyme activity difference, are these programs good enough or require developement? - Which one of them can give the best result? - And lastly, are QM/MM methods a must for an accurate answer?

I'm a high school student so sorry if my questions sound dumb.
If you can answer my inquiry's I would appreciate it:)

Thanks

**Tags:** proteins, protein-structure, proteomics, structural-biology

---

## 1 answers

### Answer by username_1 on 2021-04-16. Score: 1

Your title says `holistic`. This is a tad problematic as there's layers upon layers. Say, post-translation regulation, inhibiting metabolites, interacting protein etc. That is why when talking of an enzyme inhibitor, at the biochemical level one speaks in terms of k\_i (inhibition constant), while at the cellular level ("holistic") one talks of IC50.

This could be a biocatalysis/synthetic biology problem (make an enzyme better) or translational medicine problem (why is this mutation pathogenic).

In terms of *in silico* enzyme design, one can have all sorts of issues that need to be addressed:

* catalysis
* side reactions
* stability
* aggregation
* solubility

Also there's the expression problems, which may be due to subpar promoter, insufficient cofactors and poor codon usage.

For clinical variant assessment, a variant may:

* destabilise leaving to degradation
* remove a post-translational modification
* disrupt the active site, affecting catalysis
* lose a binding partner
* result in incorrect localisation
* result in solubility issues

So force-field scores are not the sole solution. Furthermore, a forcefield method gives one a ∆∆G (difference in relative Gibbs free energy), not a K\_M (Michaelis constant) and a k\_cat (catalytic rate), although it is somewhat possible to calculate these, or a relative variant of these.

> a variant with just 1 or 2 variants

A variant protein can have one or two *mutations* (e.g. G10R) in its sequence. A *mutant* and a *variant* are near synonyms, but variant is generally intended to be a mutant in a population.

> Does these application be used for such purpose?

For enzyme design MD simulations are not generally the first step. Rosetta pmut\_scan for example is a nice starting step as the mutational landscape of a protein is N^19, while a MD simulation takes weeks and 6+ core per single variant.
This calculates only an increase in stability (relative difference in Gibbs folding energy), not reactivity. For that you can use a transition state, whose results are inaccurate, but majorly affected by the set-up, i.e. junk-in junk-out plays a big part. There's a saying 'a week in the lab saves you an hour at the library': this is backwards for sarcasm as it happens too often, namely that is background research is always a lifesaver. An QM-MD simulation on the wild type reaction definitely can help in elucidating things if it is not clear.

## Online tools

Generally, however, there are resources out there to do many stability calculations already.

**SynBio** If you are interested in picking a residue or two to make an enzyme more stable, without resorting to a thermophilic enzyme, [PROSS](https://pross.weizmann.ac.il/step/pross-terms/) is a very nice tool.

**Clinical** (Caveat: If your variant is a variant that affects you, a friend or a relative, you/they should speak to a genetic councillor and not do research on it yourself) There are several tools that can describe a variant at the protein level. Do remember that ∆∆G is a potential so more negative is more stable —except in SDM where the score is inverted.

* [Missense3D](http://www.sbg.bio.ic.ac.uk/%7Emissense3d/)
* [Venus](https://venus.sgc.ox.ac.uk)
* [Miscast](http://miscast.broadinstitute.org/)
* [SDM](http://www-cryst.bioc.cam.ac.uk/%7Esdm/sdm.php)


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