Answers.Body: <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>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2021-04-16T12:09:51.217Z
Answers.LastActivityDate: 2021-04-16T13:35:12.523Z
Answers.LastEditDate: 2021-04-16T13:35:12.523Z
Answers.UserDisplayName: Matteo Ferla
Body: <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>
  
Tags: <proteins><protein-structure><proteomics><structural-biology>
Title: Holistic enzyme activity determination with computation
ThreadText: # 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)
  
  
  ---
  
  