## Wednesday, January 16, 2013

### Manuscript review: Mapping Enzymatic Catalysis using the Effective Fragment Molecular Orbital Method: Towards all ab initio Biochemistry

+Casper Steinmann's latest paper, which he submitted to PLoS ONE late last month, has already been reviewed!  Way to go PLoS ONE!

7. cc-pVTZ single points on one representative path should be no problem, even with MP2.  If we see only a small change in barrier I would argue against cc-pVQZ calculations.  Otherwise, we should at least do the B3LYP/cc-pVQZ calculations on the same path.

8. I don't quite follow the line and page number reference.  But we use a smaller model than
Claeyssens et al.: all atoms with 16 Å and 25 Å of the active site, respectively.  So the enzyme model is poorer.

9. Of course the reaction enthalpy in the enzyme cannot be measured, only estimated, so it's not a good benchmark.  Nevertheless, Claeyssens et al. estimate the "experimental" enzymatic reaction enthalpy to be between -15 and -13 kcal/mol. Our best prediction of the reaction enthalpy is about -6 kcal/mol (Figure 6).  Reference 37 predicts between -26 and -36 kcal/mol (and Claeyssens et al. -18.2$\pm$1.3 kcal/mol).  Clearly the predictions differ greatly, but the problem seems to lie more with the method described in reference 37.

10. I think that should actually be 5.4 kcal/mol (27.6-22.2) instead of 8.  Anyway, the main change is the inclusion of enzyme-substrate dispersion interactions, which must be weaker in the TS compared to the reactant.  It is not clear whether the largest effect is on the H-bonds to the substrate or a many small contributions from all the atoms in the blue and red region in Figure 4.  Additional calculations would be required, I don't think it's worth it.

11. Claeyssens et al. (reference 40 in our paper) computed the barrier with B3LYP/6-31G(d).

12. As we write on page 10: our barrier is 18.3$\pm$3.6 kcal/mol and Claeyssens et al.'s barrier is 9.7$\pm$1.8 kcal/mol.

13. As mentioned on page 10: The experimental activation enthalpy is 12.7$\pm$0.4 kcal/mol.  So Claeyssens et al.'s error is 3.0 kcal/mol and our error is 5.5 kcal/mol. The multilayer barrier represents just one MD snapshot.

14. This has been addressed in point 9.

From:
PLOS ONE <plosone@plos.org>
Date: 14. jan. 2013 20.56.05 CET
To: Casper Steinmann <xxx>
Subject: PLOS ONE Decision: Revise [PONE-D-12-39084]
PONE-D-12-39084
Mapping Enzymatic Catalysis using the Effective Fragment Molecular Orbital Method: towards all ab initio Biochemistry
PLOS ONE

Dear Mr Steinmann,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit, but is not suitable for publication as it currently stands. Therefore, my decision is "Major Revision."

We invite you to submit a revised version of the manuscript that addresses the points below:

There are some minor issues listed below, but one primary problem that must be addressed with additional text and possibly the single point calculations that the referee refers to in point 7. The serious energy discrepancy must be addressed as amply discussed by the reviewer.
We encourage you to submit your revision within forty-five days of the date of this decision.

When your files are ready, please submit your revision by logging on tohttp://pone.edmgr.com/ and following the Submissions Needing Revision link. Do not submit a revised manuscript as a new submission. Before uploading, you should proofread your manuscript very closely for mistakes and grammatical errors. Should your manuscript be accepted for publication, you may not have another chance to make corrections as we do not offer pre-publication proofs.

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PLOS ONE

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Reviewer's Responses to Questions

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #1: (No Response)

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #1: (No Response)

3. Does the manuscript adhere to standards in this field for data availability?

Authors must follow field-specific standards for data deposition in publicly available resources and should include accession numbers in the manuscript when relevant. The manuscript should explain what steps have been taken to make data available, particularly in cases where the data cannot be publicly deposited.

Reviewer #1: Yes

Reviewer #1: (No Response)

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors below.

Reviewer #1: Yes

Reviewer #1: (No Response)

dual publicationor research or publication ethics.

Reviewer #1: This paper describes the implementation and first tests of the frozen-domain approximation for the effective fragment molecular orbital method for the chorismate mutase enzyme reaction. The calculations are carefully tested and well done. The results are interesting and therefore publishable. However, compared to previous calculations, the approach shows very large deviations in some energies. These need to be explained before the method can be trusted (points 9 and 10).
1. The level of EFMO should be specified in the abstract.
2. It should be shortly discussed how dangling bonds in the QM calculations are treated.
3. The protonation of all residues should be specified, in particular for the His residues. The number of counter ions and their nature (what element?) should also be specified.
4. I suppose Bohr should also be written with a capital B.
5. The legend of Figure 3 should point out that C1 and C5 is the shared atom.
6. I suppose the references to Figure 2 on p. 6 are misprints (should be Figures 4 and 5).
7. Single-point calculations should also be performed for isolated chorismate with TZP and QZP basis sets, both at B3LYP and MP2 levels of theory, to check the basis set effects.
8. Line -5 on p. 9: Is the enzyme model poorer in the present or the previous study?
9. The ~30 kcal/mol difference in the reaction energy between the present results and those in refs 36 and 37 is very alarming. It sounds unlikely that this should be caused by the restricted relaxation in this study (the large model changes the energy by only 3 kcal). This difference must be better explained. How could the authors expect that we should trust a method that can give errors of 30 kcal?
10. Why do the multilayer calculations give so different results (8 kcal) compared to the ONIOM calculations? Which approach is best? Again, this large difference is very alarming and reduces the credibility of the present approach.
11. Still another reason for the difference between the present results and that by Claeyssens et al. is of course the use of LCCSD(T0) with large basis sets in the latter study.
12. Is the energy spread among the 7 snapshots similar or larger than in previous studies?
13. I cannot see that a doubling of the computational cost is a big problem. On the other hand, in my eyes a method that gives errors of 30 kcal, or even 8 kcal is completely useless. The authors should think more on the accuracy than on the timing.
14. Is the reaction enthalpy experimentally known? It seems to be harder to estimate than the activation enthalpy and therefore more interesting to study and reproduce.

6. If you would like your identity to be revealed to the authors, please include your name here (optional).

Your name and review will not be published with the manuscript.

Reviewer #1: (No Response)