Review Reports - Nonenzymatic Deamidation Mechanism on a Glutamine Residue with a C-Terminal Adjacent Glycine Residue: A Computational Mechanistic Study

Round 1

Reviewer 1 Report

In this piece of research the authors perform a computational mechanistic study of nonenzymatic glutamine deamination. This study is strongly related to that of Ref. 28 from the same research group. I find these results interesting, the paper is well written, and therefore I recommend its publication. Two minor changes are suggested.

Since the paper is related to Ref. 28 and some results are compared to those from that paper, I believe that, for the sake of clarity, the equivalent of Figure 2 for the Ac-Gln-NMe system is advised to be presented in the manuscript.
The highest level of calculation employed, MP2/6-311+G(2d,2p) is usually not accurate enough to describe barrier heights. Bbenchmark calculations using additional DFT and ab initio levels are suggested to corroborate the obtained barrier height values.

Author Response

Reviewer 1

Comment:

Since the paper is related to Ref. 28 and some results are compared to those from that paper, I believe that, for the sake of clarity, the equivalent of Figure 2 for the Ac-Gln-NMe system is advised to be presented in the manuscript.

Response:

We appreciate the reviewer’s advice. The optimized geometries of the cyclization step for Ac-Gln-NMe system was added as Figure 5.

Comment:

The highest level of calculation employed, MP2/6-311+G(2d,2p) is usually not accurate enough to describe barrier heights. Benchmark calculations using additional DFT and ab initio levels are suggested to corroborate the obtained barrier height values.

Response:

We understood the importance of benchmark calculations and performed the additional calculations with CAM-B3LYP/6-311+G(2d,2p), ωB97XD/6-311+G(2d,2p), and HF/6-311+G(2d,2p). The obtained relative energies were shown in Tables 4 and 5. In addition, the below sentences were added to Line 246–256.

Added sentences:

To compare the activation barrier of the reaction in Ac-Gln-NMe and Ac-Gln-Gly-NMe more accurately, the activation barriers were calculated for both model compounds using the B3LYP/6-31;G(d,p), MP2/6-311+G(2d,2p), CAM-B3LYP/6-311+G(2d,2p), ωB97XD/6-311+G(2d,2p), and HF/6-311+G(2d,2p) levels (Tables 4 and 5). The energy obtained by HF methods was too high for the reaction proceeding. MP2 methods yielded the lowest activation barrier, and the activation barriers in CAM-B3LYP/6-311+G(2d,2p) and ωB97XD/6-311+G(2d,2p) were similar to that in B3LYP/6-31+G(d,p). The MP2/6-311+G(2d,2p) level has been used to investigate the activation barrier of nonenzymatic posttranslational modification of proteins, and the calculated values were reasonable in comparison with the experimental values [36,38]. Therefore, the energy diagram in this calculation level was shown in Figure 10.

Reviewer 2 Report

The authors have built upon their previous study on glutamine deamidation (Ref 28) and performed a similar investigation in the current manuscript, with the main difference being the addition of a (N+1) Gly to their computational system. They found an additional hydrogen bond with Gly as the major reason for the “accelerated” deamidation rate. This would be a reasonable addition to the authors’ efforts toward computational analyses of non-enzymatic reactions involving asparagine and glutamine, but some improvements must be made before this manuscript can be published.

First, is there experimental evidence showing deamidation of Ac-Gln-Gly-NMe is indeed faster than that of Ac-Gln-NMe? If not, I believe the authors should EITHER avoid using the term “Acceleration” (and change the title and wording to something similar to their previous study in ref 28) as it implies a matter of fact OR compare their computational results of Ac-Gln-Gly-NMe with another sequence that has relevant experimental data.

Second, the comparison between Asn and Glu can be made more clear. A schematic diagram might be presented to highlight the structural differences between Asn and Glu deamidation pathways. If the authors felt these 2 are too similar to be worth of illustrating, they should at least speculate the structural reasons for the much slower deamidation rate of Gln. In addition to the comparison of structures, the comparison of energy profiles needs more details. Although the authors did cite and compare computational activation barriers at line 242, it is not clear if these referred studies used similar QM methods and/or structures.

Third, the authors should briefly explain why they didn’t perform similar calculations on the Gln-Trp sequence, which could serve as a very good example of higher activation energy (corresponding to lower reaction rate) than the Gln-Gly sequence. In addition, I hope the authors can provide some details as to how they conceived the 3 initial conformations for pathways A, B, and C.

Fourth, it would help readers to better understand the repositioning of the H2PO4- ion if a figure can be made for the structural illustration of INT1 to INT2.

Other minor issues:

Spell out all acronyms rather than only for reactant complex (RC).

Scheme 1 not referred to in the main test?

Line 244 should refer to Figure 8 instead of Figure 4?

Some grammatical problems such as line 71 “supposed” might be “proposed”, lines 119 to 120, line 257 “pathway” should be “pathways”, line 266 “the side chain peripheral residues does not obstacle to” might be “side chain of peripheral residues does not impede” etc.

Author Response

Reviewer 2

Comment:

Response:

We appreciate the reviewer’ comment. There are no experimental data showing that the deamidation rate of Ac-Gln-Gly-NMe is faster than that of Ac-Gln-NMe because the experimental data for Gln deamidation was obtained using pentapeptide. The experimental data for pentapeptides only show that deamidation is fastest when the (N + 1) residue is Gly. Therefore, we modified the title to “Nonenzymatic deamidation mechanism on a glutamine residue with a C-terminal adjacent glycine residue: A computational mechanistic study”.

Comment:

Response:

In pathway A and B, although almost no changes of dihedral angle φ were observed in TS1 formation, ψ was changed by 37° (pathway A) and 47° (pathway B). In contrast, φ and ψ were changed by 14° and 31° in Asn deamidation [Ref.38]. The proton transfer and hydrogen-bond formation related to TS1 formation were similar. Therefore, the conformational changes of the main chains are thought to be major difference between Asn and Gln deamidation. The difference in the length of the side chain affects the conformational changes. As the result, twisting of the main chain may occur, and TS1 may be destabilized in Gln.

The compared activation barriers include the values obtained using B3LYP/6-31+G(d,p) or MP2/6-311+G(2d,2p), and no similar structures were used because of the studies for Asn deamidation. However, the activation barriers obtained by the same calculation level were 80-90 kJ/mol [Ref.38].

We added the below sentences to clarify these as well.

Added sentences to Line 276:

The optimized geometries proposed the reason why the activation barrier of Gln deamidation was higher, or slightly higher, than that of Asn deamidation. In Asn deamidation, the structural changes of the main chain for the TS1 formation occurred to on both N- and C- terminal sides [38]. On the other hand, TS1 formation in Gln deamidation required the structural changes of the main chain only on the C-terminal side. Since the side chain of Gln is longer, the conformational change upon cyclization occurs in the side chain and does not affect the main chain on the N-terminal side. As the result, twisting of the main chain may occur, and TS1 may become unstable.

Added sentences to Line 265:

In addition, these activation energies include values obtained at the same calculation level as that in the present study. The calculated activation barrier of Gln deamidation was higher or slightly higher than that of Asn deamidation.

Comment:

Response:

We apologize the lack of explanation and the impossibility of a brief explanation. In general, deamidation rates of Asn and Gln are affected by the bulkiness of the side chain on the (N + 1) residue. In the Asn-Pro sequence, since the cyclic intermediate cannot be formed, the deamidation rate of Asn is slowest. The deamidation occurs by direct hydrolysis of the side chain in the Asn-Pro sequence. This mechanism is applied to Gln deamidation, and the deamdation rate in Gln-Pro is same as that in Asn-Pro [Ref.25]. However, the deamidation rate of Gln is slowest when the (N + 1) residue is Trp. This suggests that the cyclic intermediate cannot be formed in Gln-Trp sequence and the direct hydrolysis of the side chain is unlikely to occur. Unknown mechanism may underlie. In addition, the Gln-Phe sequence has the smallest damidation rate in faster than the deamidation of the Gln-Pro sequence, whereas the (N + 1) residue of the most deamidated Gln residue in γS-crystallin is Phe [Ref. 8]. For above reasons, there are many challenges for sequences with slow deamidation rates, which we hope to clarify the mechanisms in the future. Since explaining this fact would be redundant, we added briefly some of these reasons to the manuscript.

RC-A, -B, and, -C were constructed based on the optimized geometry of RC in Ac-Gln-NMe [Ref. 28]. When the conformation is almost not changed, RC is stabilized by the increase of hydrogen bonds between the catalytic ions and peptides [Ref. 35]. Therefore, we examined positions of the H₂PO₄⁻ ion which can form two hydrogen bonds involved in the proton transfer and one with the main chain. As the results, RC-A, -B, and, -C were obtained. We added these sentences to Line 129.

Added sentences:

Line 69: Glutarimide formation is assumed not to occur in sequences with slower deamidation rate than Gln-Pro sequence.

Line 315: Many mysteries in the slow deamidated sequences, such as Gln-Trp, will be solved in the future studies.

Comment:

Fourth, it would help readers to better understand the repositioning of the H₂PO₄^- ion if a figure can be made for the structural illustration of INT1 to INT2.

Response:

We appreciate the great proposal. The structural illustration of conversion from INT1 to INT2 was added as Figure 8.

Other minor issues:

Comment:

Spell out all acronyms rather than only for reactant complex (RC).

Response:

For all abbreviations, I apologize for not knowing where they are spelled out. All acronyms were included in the caption of Scheme 2. In addition, we added the below sentences to Line 122-125.

Added sentences:

The optimized geometries of reactant complex (RC), transition state 1 (TS1), and intermediate 1(INT1) for the cyclization step were obtained, and the optimized geometries of intermediate 2 (INT2), transition state 2 (TS2), and product complex (PC) for deammoniation step were obtained.

Comment:

Scheme 1 not referred to in the main test?

Response:

We apologize for the lack of citation for Scheme 1. In the revised manuscript, it is referred in Line 61.

Comment:

Line 244 should refer to Figure 8 instead of Figure 4?

Response:

Thank you for pointing out our mistake. The figure number was corrected.

Comment:

Response:

We apologize for the grammatical problems. The entire text has been reviewed and revised.