Thermodynamics and Kinetics of Glycolytic Reactions. Part II: Influence of Cytosolic Conditions on Thermodynamic State Variables and Kinetic Parameters
, Hauke Harms 1 and Thomas Maskow 1,*
Round 1
Reviewer 1 Report
The authors tested two theoretical models in describing the experimentally determined effects of physiologically relevant variations in conditions on the thermodynamics and kinetics of the reactions catalyzed by enolase and phosphoglucose isomerase. The results favor a flux-force model in kinetic description. Although the findings may seem trivial, they provide a useful addition to what is known about these reactions because they are related to cell conditions, i.e. cellular concentrations of low-mass effectors and macromolecules causing crowding effect. My comments refer mainly to technical problems.
- The title of the manuscript indicates that it is “Part II”. It is not immediately clear from the text what was Part I, so that the reader could follow the evolution of ideas, approaches, etc? In addition, the title seems unfinished – properties of what? For brevity, one could just omit “on thermodynamic and kinetic properties”.
- There seems to be an overlap in the enolase data with a seemingly parallel publication (ref 6, printed version to appear in October 2020). The authors should check and provide appropriate citing instead of duplication, if any.
- The range of the Mg2+ concentrations used (1-15 mM) does not cover both sides of the expected cellular free Mg2+ range (0.5-1 mM). 15 mM may be too much.
- “Gibbs energy” in place of “Gibbs free energy” or even “Gibbs free energy change” looks like a lab jargon. The same refers to the use of “enthalpy” and “entropy”.
- Expressions like “as described in [ref]” seem inappropriate as reference by itself cannot be a clause member. Language editing is needed in some places.
Author Response
Please see the attached file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Title: Thermodynamics and kinetics of glycolytic reactions. Part II: Influence of cytosolic conditions on thermodynamic and kinetic properties
Authors: Kristina Vogel, Thorsten Greinert, Monique Reichard, Christoph Held, Hauke Harms, Thomas Maskow
The authors of the manuscript have examined the influence of cytosolic media on the glycolytic reactions from thermodynamic and kinetic properties.
They made an attractive and comprehensive study comparing the classical method in a more realistic environment. It is remarkable that the authors examining the solvent effect of this reaction using a very complicated media (cytosol). The fundamental problem of the solvent effect has been studied in numerous articles previously using simple solvents such as water or simple buffered solvent mixture. Analogue studies were also investigated previously in the field of chemistry, where the complex effect of the entire media was considered, such in
1. J. Am. Chem. Soc. 2005, 127, 7615; doi.org/10.1021/ja042227q);
2. F. Ruff; I.G. Csizmadia; „Organic Reactions: Equilibria, Kinetics and Mechanism” ISBN-13: 978-0444881748;
3. C. Reichardt, T. Welton „Solvents and Solvent Effects in Organic Chemistry” ISBN-13: 978-3527324736). These publications could be cited here as well in the introduction to highlight the topic.
They presented nice results on the reversibility of this bioreactions. Their hypothesis on reversibility was tested by two models, Noor’s and the flux-force model. Each method shows similar trends, supporting the concept.
The Materials and Method section is correct. The figures are understandable but not too attractive. The massage and the information did not arrive on the first sight. In general, the quality of the figures could be improved.
I found the manuscript clear and well written; the context is well exposed.
The present results are suitable to be published after some minor corrections.
I recommend accepting the publication of this manuscript in the journal.
Some points to be addressed
1. I suggest to put the reaction into the first possible row of the paper, it could be highlight in the first page, in order to clarify the topic. I suggest to extend these reactions by the name of the enzyme, the environment, the conditions.
2. The introduction is very limited, it can be extended with a few sentence about solvents and solvent effect in general and the point of the G6P transformation briefly. Suggested references are in the first paragraph.
3. In figure 4, the black points follow a reasonable fit to a linear function if the first few points (between 0.015 and 0.018) are omitted from the fitting. This modification would not change very much the fitting parameters, but the reliability would be better. I suggest listing the fitting parameters (black and red) within the Figure itself, in order to make easier the results for the reader.
Line 37: “It is therefore necessary” -> “It is therefore, necessary”
Line 1: “lowerd” -> “lowered”
Line 467: “flux-force model show” -> “flux-force model shows”
Line 468: “In addition both models” -> “In addition, both models”
Line 470: “Noor's model provide neither” “Noor's model provides neither”
Author Response
Please see the attached file.
Author Response File: Author Response.pdf
Reviewer 3 Report
The review of the paper “Thermodynamics and kinetics of glycolytic reactions. Part II: Influence of cytosolic conditions on thermodynamic and kinetic properties”
The paper aims at the study of thermodynamic and kinetic effects introduced by molecular crowding and high ionic strength as a model of conditions in the cytosol. First I must admit that it was a very demanding review for me, to some extent due to the authors' sloppiness in the submission of the paper. As a result, it took me more time to do the review.
The authors send the second part of their study with a part I still not accepted and did not find it sensible to attach it to the submission. I felt as if reading a second part of the book. For the future, I advise the authors more ‘reviewer friendly’ approach.
Generally, as an enzymologist and person using from time to time ITC to study enzyme kinetics, I found these papers (part I and part II) quite interesting. Admittedly it is not easy to read as Noor and force-flux models are not yet frequently used in enzymology. Part II is not a standalone piece.
I have marked multiple problems of the paper (in the terms of clarity) in the attached marked manuscript. Here I will address several major reservations which will be better addressed in the text:
The unclear description of the ITC experiment
The ITC experiment is described in a very unclear way. First of all the authors describe in4.3 the very slow injection of the F6P to the enzyme. The reasons for such extended injection (over 60 min) are not provided and this leaves kinetic analysis in a shady light.
This is in contrast to the procedure used in [6] and the description of the enolase reaction. There the reaction was initiated with one quick (app. 80s) injection of the enzyme to the reagent volume.
Also, no information on the reference measurements are provided (was it enzyme to buffer, inactivated enzyme to substrate? Or in the first case buffer to an enzyme).
This is important as with different viscosities and ionic strength this may have influenced the results.
My confusion is more extensively described in the comment.
Also the description of the way the authors calculated reaction conversion (end concertation of reagents) is very very difficult to grasp. One has to have a deep knowledge of the ITC inserts and this particular reaction to understand that just evacuation of the reaction vessel and killing the enzyme by acid or ACN is not yielding the good measure of the product using LC-MS. Especially the whole story of calculating the equilibrium is very strange (I would just do the experiment outside the calorimeter, waited for equilibrium and sampled with a sensitive technique which immediately kills the enzyme). One can see that authors did invest a lot of thought into the subject but it is very difficult to grasp this even for somebody who did similar experiments himself.
Unconvincing explanation of changes of enthalpy
The discussion on observed changes in reaction enthalpy (page 13) in the function of different conditions is very unconvincing. Several disjointed ideas are presented there and I doubt that the authors have a specific hypothesis which explains why there is an increase of enthalpy upon the addition of PEGs. I cannot agree that this is due to volume exclusion as this is associated with a potential change of system entropy. This is clearly stated in the manuscript yet somehow authors claim it influenced enthalpy (The interaction of these 3 effects could explain the effect of crowding on the reaction enthalpy.). There are also several speculative statements on the potential interaction of PEGs with something (not mentioned if with reagents, enzyme?) but I still do not understand how any change in the activity of the enzyme can result in a change of reaction enthalpy? It can influence equilibrium and therefore DG of the system but how it influences the DH of the reaction eludes me. Also, the authors try to explain the observed effects by a change of dielectric constant while they do not observe a significant effect of ionic strength (and ion screening effect is known to eliminate electrostatic interactions and effectively decrease dielectric constant).
I will not mention heresy about BSA not interacting with other proteins… It must be some edition error and I do not think the authors truly meant it.
Summing up this section is very weak and needs improvement and more thought from the authors.
Finally, there is an issue with the paper [6]
I would like to ask for an explanation what is the content relation between the paper in BBA - General Subjects 1864 (2020) 129675 and this under review (as the reaction 9 is also under consideration in cytosolic condition) and specifically fig 6 in [6] and fig. 8 in the paper under consideration. These two figures are identical (although the former presents DrH in the function of the studied parameters and the latter provides kinetic constant L). All the trends are the same and of course, the scale is different due to different parameters under evaluation and different types of concentration (g /kg vs mmol/kg for PEG). However one can see the data are the same. This is at least a reprint of the same data.
It surprises me that the same trends and values are obtained for thermodynamic and kinetic parameters.
Finally, I would like to suggest that this article would be of much higher value if it was accompanied by the data deposition in a repository (like Mendeley data).
It would be very instructive to provide raw calorimetric data with reference experiments and then integrated data giving Cs(t) and P(t) and finally data used for the kinetic fit. I would certainly test the described method first on the provided data and then on my experiments.
Comments for author File: Comments.pdf
Author Response
Please see the atached file.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
The authors conducted a comprehensive revision of their manuscript and provided convincing arguments to my remarks. I think the paper is now ready for publication.
