Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine-Residue Catalyzed by Dihydrogen Phosphate Ion Tomoki Nakayoshi, Koichi Kato, Eiji Kurimoto, and Akifumi Oda J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10386 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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The Journal of Physical Chemistry

Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine-Residue Catalyzed by Dihydrogen Phosphate Ion

Tomoki Nakayoshi†,‡, Koichi Kato†,§, Eiji Kurimoto†, Akifumi Oda†,¶,*

†Graduate

School of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya,

Aichi 468-8503, Japan ‡Institute

of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-

machi, Kanazawa, Ishikawa 920-1192, Japan §Department

of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya,

Aichi 463-8521, Japan ¶Institute

for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

*Corresponding Author: [email protected], +81-52-832-1151

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Uncommon crosslinked amino acids have been identified in several aging tissues, and are suspected to trigger various age-related diseases. Several uncommon residues are formed when the dehydroalanine (Dha) residue undergoes a nucleophilic attack by surrounding residues. Dha residues are considered to be formed by post-translational modification of serine (Ser) and cysteine residues. In the present study, we investigated the Dha-residue formation mechanism catalyzed by dihydrogen phosphate ion (H2PO4−) using quantum chemical calculations. We obtained optimized geometries using B3LYP density functional method, and carried out singlepoint energy calculations using second-order Møller-Plesset perturbation method. All calculations were performed using Ace-Ser-Nme (Ace=acetyl, Nme=methylamino) as a model compound. Results of computational analysis suggest that the mechanism underlying the Dharesidue formation from Ser consists of two steps: enolization and 1,3-elimination. The H2PO4− catalyzed both reactions as proton-relay mediator. The calculated activation barrier for Dharesidue formation was estimated as 30.4 kcal mol−1. In this pathway’s reactant, the catalytic H2PO4− interacts with the Ser-residue α-proton, carbonyl oxygen of Ser and C-terminal side adjacent residues, and the calculated activation energy was reproduced the experimentally reported value for nonenzymatic modifications of amino acid residues. Therefore, our calculation suggests that H2PO4−-catalyzed Ser-residue dehydration can proceed nonenzymatically.


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1.

INTRODUCTION

In recent years, amino acid residues that have undergone nonenzymatic modifications have been detected in proteins in aging tissues. Nonenzymatic modifications of amino acid residues include glycation1‒4, stereoinversion5‒9, deamidation10‒13, and cross-linking14‒17, and these chemical modifications frequently cause protein degradation. These modified amino acids tend to accumulate in long-lived proteins. Crystallin is one of the most long-lived proteins in vivo. Crystallin protein contained in the human lens; it is almost completely formed at birth, and shows minimum turnover throughout the life18,19. Therefore, crystallin is frequently used for studies of age-dependent amino acid modifications. The accumulation of abnormal amino acids causes aggregation and insolubilization of crystallin, which triggers cataract20,21. Thus, the lens of cataract patients contains a lot of undesirable modified amino acids compared with that of healthy individuals. Serine (Ser) is a relatively reactive amino acid and undergoes various chemical modifications. However, only a few studies have examined the detailed mechanism of nonenzymatic modifications of Ser. Ser is prone to stereoinversion in peptides and proteins. DSer residues are detected in α-crystallin in the eye lens and in β-amyloid in brain8,9,22,23. Hooi et al. reported an age-dependent accumulation of D-Ser residue, and showed that Ser59 and Ser62, which are located in an unstructured region of the αA-crystallin23, undergo stereoinversion. Although the Ser-residue stereoinversion mechanism has not yet been elucidated in detail, Lyons et al. have suggested that Ser residues undergo stereoinversion by simple α-proton abstraction24. Takahashi et al. reported the Ser-residue stereoinversion mechanism catalyzed by two water molecules or dihydrogen phosphate ion (H2PO4−) using quantum chemical calculation25,26, and predicted that the Ser residue tends to be stereoinverted more frequently than other amino acid



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residues, except aspartic acid (Asp), because the transition state of α-proton abstraction is stabilized by negative hyperconjugation. Another example of abnormal modification of the Ser residue is covalent crosslinks14‒17. The formation of crosslinks has been identified in food proteins, and has long been studied from a nutritional point of view27‒30. Treatment of protein-containing foods with heat and/or alkali results in the crosslinking of amino acid residues. O

O OH

O N H

L-Ser

HN

NH

S

H N

HN

O

O

NH

O

Lanthionine (Lan)

+ Cys O OH

O N H

Enolized Ser

‒ H2O H N

OH

+ H2O

O

H N

N H

+ His

HN

O HN

Dha

O

O NH

Histidinoalanine (Hal)

D-Ser

O

OH

N H

N H

O

+ Lys O

N

N

H N

O

HN

O

HN

N H O

NH

H N O

Lysinoalanine (Lal)

Scheme 1. Formation mechanism of abnormal crosslinked amino acid residues via dehydroalanine (Dha)

The formation of crosslinks reduces the nutritional value of the protein because it reduces some amino acid content. Hydroxide anions (OH−) in alkali-treated system are considered to assist these crosslinking reactions15,27. Crosslinked amino acid residues have also been identified in aging tissues, which are believed to be formed via the dehydroalanine (Dha) residue produced


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via post-translational modifications of Ser and cysteine (Cys) residues14‒17,27,31,32. Srivastava et al. analyzed αA-crystallin in aged eye lens, and reported the conversion of Ser59 to Dha33. Dha residues have been reported to be relatively stable in the absence of nucleophiles24, and have also been identified in the crystal structure of several proteins34,35. However, Dha residue easily reacts in the presence of nucleophiles such as Cys, histidine (His), and lysine (Lys) residues resulting in the formation of lanthionine (Lan), histidinoalanine (Hal), and lysinoalanine (Lal), respectively14,27. Scheme 1 shows the mechanism of formation of Lan, Hal, and Lal residues via Dha. The crosslinks formed in these reactions are presumed to cause a significant change in the flexibility of proteins. Crosslinked amino acids are detected not only in the lens but also in aorta and dentin31,32,36,37. The α- and β-carbons of Dha residue are sp2 hybridized. Therefore, if a water molecule nucleophilically attacks the β-carbon of Dha residue, both L- and D-Ser residues are formed. Thus, Dha residues can potentially act as intermediates for the stereoinversion of Ser residues (Scheme 1). However, the Dha-residue formation mechanism has not been elucidated in detail. Since there is no significant amount of OH− at physiological condition (pH 7.4), it is unlikely that Dha residues are formed in living body via the same mechanism as in the alkali treatment of food protein. Ser-residue dehydration would have to be catalyzed in order to proceed in vivo. The present study aimed to investigate the mechanism of Dha-residue formation, catalyzed by inorganic phosphate species, abundant in the living body. Although, inorganic phosphate species comprise four types of charge states (H3PO4, H2PO4−, HPO42−, and PO43−), under physiological conditions only the H2PO4− and HPO42− are present at significant concentrations, since the pKa values of H3PO4, H2PO4− and HPO42− are 2.15, 6.82, 12.38, respectively. Therefore, H2PO4− was used as a catalyst acting as both Brønsted acid and base under physiological conditions.



2.

THEORETICAL METHODS

All calculations were performed using Gaussian 1638. Because the computational cost of quantum chemical calculation is very large, it is impossible to calculate the entire protein without any special efforts. In this study, all the calculations were performed using a model compound shown in Figure 1, in which a Ser residue was capped with an acetyl (Ace) and a methylamino (Nme) groups on the N- and C-termini, respectively. Conventionally, amino acids capped with Ace and Nme groups are used for the determination of properties of amino acid residues in peptides and proteins39,40. The dihedral angles φ (C-N-Cα-C) and ψ (N-Cα-C-N) characterize the main-chain conformation, and the dihedral angles χ1 (N-Cα-Cβ-Oγ) and χ2 (Cα-Cβ-Oγ-H) characterize the side-chain conformation. However, since the Dha residue does not harbor Oγ in the side-chain, the χ1 and χ2 dihedral angles are not defined for Dha. Previous studies41‒43, have performed geometry optimization for nonenzymatic modifications of amino acid residues using B3LYP density functional methods, and obtained calculation results consistent with experimental ones. For comparison, we used the same level of theory as the previous papers in this study. Therefore, energy minima and transition-state (TS) geometries are optimized without any constraints by density functional theory (DFT) calculations with the B3LYP exchange-correlation functional and the 6-31+G(d,p) basis set. All the geometry optimizations were performed in the aqueous phase, using polarization continuum model (PCM) calculations based on field methods of solvent reaction. The PCM used default settings in Gaussian 16, and the dielectric constant of water was set to 78.355. The hydration free energies were calculated by subtracting the electronic energies in the gas phase from those in the aqueous phase, which were obtained from a single-point calculation for the optimized geometries


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in the aqueous phase. The vibrational frequency calculations were performed at the B3LYP/631+G(d,p) level of theory for all optimized geometries to confirm them as energy minima (with no imaginary frequency) or TSs (with a single imaginary frequency), and to obtain the zero-point energy (ZPE). Intrinsic reaction coordinate (IRC) calculations were performed at the B3LYP/631+G(d,p) level of theory from the TSs, followed by full geometry optimizations, to confirm that each TS connects two energy minima. Moreover, to obtain more precise energy values, singlepoint energy calculations were performed using the second-order Møller-Plesset perturbation theory (MP2) calculation with the 6-311+G(d,p) basis set at all the optimized geometries. All the single-point electronic energies of energy minima and TS geometries were calculated at the MP2/6-311+G(d,p) level of theory and further corrected for the ZPEs and the Gibbs energies at 1.00 atm and 298.15 K.

O

H 3C

γ

χ2 β

OH χ1

φ

α

N H

ψ

H

H N CH3

O

Figure 1. Structure of the model compound Ace-Ser-Nme used in this study. The dihedral angles φ (C-N-Cα-C) and ψ (N-Cα-C-N), which characterize the main-chain conformation, and dihedral angles χ1 (N-Cα-Cβ-Oγ) and χ2 (Cα-Cβ-Oγ-H), which characterize the side-chain conformation, are indicated.

3.

RESULTS AND DISCUSSION

3.1. 1,2-Elimination pathway



It is presumed that Dha residues can be formed by 1,2-elimination of Ser (Scheme 2). During the 1,2-elimination process, the abstraction of the Ser-residue α-proton and the elimination of the Ser-residue side-chain OH group lead to one-water-molecule elimination. Catalytic H2PO4− was placed around the model compound and the reactant complex E-RC was prepared. Figure 2 shows the optimized geometries of the 1,2-elimination system. During this reaction, the E-RC complex was converted into E-PC via E-TS. In the E-RC, three interactions were formed between the H2PO4− and the model compound, including the CαHα−O interaction and two hydrogen bonds. The distance of the CαHα−O interaction is 2.281 Å. The 1,2-elimination process continues by double proton relays via H2PO4−. In the E-TS, the water-molecule elimination proceeds and the abstraction of Hα by catalytic H2PO4− is nearly complete. E-TS has a single imaginary frequency of 745i cm−1 (i: imaginary unit). During the conversion of E-RC to E-PC, φ and ψ change by 22° and 11°, respectively. Although during the 1,2 elimination process the conformational change of the main chain is relatively minor, the formation of E-PC from E-TS2 is associated with an extremely large migration of the catalytic H2PO4− and one water molecule. Based on the E-RC, the estimated activation barrier of the 1,2-elimination process was 35.4 kcal mol−1, which is too high to achieve under physiological conditions. In general, during the 1,2elimination process, the Cα-Hα bond should be antiperiplanar to the cleaving Cβ-Oγ bond. However, given the low leaving ability of the hydroxyl (OH) group 1,2-elimination requires that the Ser-residue side-chain OH group is protonated. In other words, when only H2PO4− acts as a catalyst, the Cα-Hα bond cannot be antiperiplanar to the cleaving Cβ-Oγ bond. One possible cause for the high activation energy may be that the TS cannot be sufficiently stabilized.


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OH

O N H H

H N O

− H2O + H2O

O N H

H N O

Scheme 2. Dha-residue formation mechanism from Ser by 1,2-elimination

E-EN1

E-TS

E-RC

1.746 1.855 1.667 1.792

2.281

2.096

1.178

1.709

1.233

0.988

1.734

Figure 2. Calculated geometries of the 1,2-elimination system: E-RC (φ = −158°, ψ = 179°, χ1 = −165°, χ2 = −162°), E-TS (φ = −152°, ψ = −179°, χ1 = −103°, χ2 = −125°), and E-PC (φ = 180°, ψ = −169°, χ1 and χ2 are not defined). The selected interatomic distances are shown in Å.

3.2. Pathways via enol tautomer Although we investigated the pathway of Dha-residue formation by 1,2-elimination, its activation energy (35.4 kcal mol−1) is somewhat higher for the process to occur under physiological conditions. Therefore, we identified the Ser-residue dehydration pathways via the enol tautomer (Scheme 3). Enol tautomer dissociated to form a Dha residue and one water molecule by 1,3-elimination. Elimination via the enol tautomer is a common organic chemical reaction observed in aldol condensation44. In the present study, we used two types of conformers (R1 and R2) for calculation (Figure 3). Takahashi et al. have previously studied the enolization of the Ser-residue using an R1 conformer26. R1 is stabilized by the OH group in the side-chain of



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Ser residue, which interacts with adjacent residues and is unique to Ser residues. R2 is a conformer that is 0.33 kcal mol−1 more stable than R1 according to DFT calculations, however, the phosphate-catalyzed mechanism of Ser-residue enolization using R2 remains to be investigated, and no previous studies have reported the Dha-residue formation mechanism from enolized Ser residues. We placed H2PO4− around the optimized model compound, and prepared three types of reactant complexes (1-RC, 2-RC, and 3-RC), which naturally connect Ser and Dha residues. Although both 1-RC and 2-RC are composed R1 and H2PO4−, the model compound interacts with H2PO4− in different ways. The 1-RC is approximately 1.81 kcal mol−1 more stable than 2-RC, and the energy of 3-RC, which is composed R2 and H2PO4−, is almost equal to that of 1-RC. In the gas phase, although the electronic energy of 1-RC was slightly more stable than that of 2-RC by 1.70 kcal mol−1, there was no substantial difference between them; by contrast, 3-RC was far more unstable than 1-RC and 2-RC, and its electronic energy was higher than that of 1RC by 24.5 kcal mol−1. Thus, it appears that 3-RC is highly stabilized by hydration effects.

OH

O N H H

OH

O

H N

N H

O

− H2O

H N

+ H2O

OH

O N H

H N O

Scheme 3. Dha-residue formation mechanism from Ser comprising two steps: enolization and 1,3-elimination.

R1 1.729

1.960

R2 2.536


2.011

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Figure 3. Optimized conformers of the model compound (Ace-Ser-Nme) used in this study. Selected interatomic distances are shown in Å.

3.2.1. Pathway 1 In the enolization process, H2PO4− abstracts the α-proton of the Ser residue, and a proton from H2PO4− is passed to the main-chain amide oxygen of the Ser residue. Figure 4 shows the optimized geometries of the enolization system. In this reaction, 1-RC is converted to 1-EN1 via 1-TS1. In 1-RC, three interactions are formed between the H2PO4− and model compound including CαHα−O interaction and two hydrogen bonds. The distance of CαHα−O interaction is 2.347 Å. Enolization proceeds by double proton relays via H2PO4−; however, these proton transfers are asynchronous. The geometry of 1-TS1 is rather similar to that of 1-EN1 than 1-RC, and the abstraction of Hα by catalytic H2PO4− is almost complete. 1-TS1 has a single imaginary frequency of 1037i cm−1 (i: imaginary unit). During the conversion of 1-RC to 1-EN1, φ and ψ change by 12° and 33°, respectively. In this process, the length of hydrogen bonds formed between Ser side-chain OH group and the adjacent-residue main chain is greatly extended. This attenuation of interaction is presumed to weaken the constraints of side-chain conformation, and facilitates the formation of the reactant of 1,3-elimination process (1-EN2). Based on 1-RC, the activation barrier of the enolization process was estimated as 23.3 kcal mol−1. 1-EN2 is formed by the rotation of the Ser-residue side chain of 1-EN1. Rotation of the Ser-residue side chain is a three-step process with insignificant changes. Figure S1 of the Supporting Information presents the energy minima and TS geometries found during the rotation of the Ser-residue side chain. The difference between the values of χ1 of 1-EN1 and 1-EN2 is



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164°. When 1-EN2 is yielding, new hydrogen bonds are formed between the Ser-residue sidechain OH and H2PO4−. In the 1,3-elimination process, H2PO4− abstracts the OH proton of the enolized Ser residue and passes its own proton to the Ser-residue side-chain OH group. Figure 5 shows the optimized geometries of the 1,3-elimination system. In this process, 1-EN2 is converted to 1-PC via 1-TS2. During this conversion, double proton relays via H2PO4−, and the Cβ‒Oγ bonds of the Ser-residue side chain are cleaved. Although the distance of Cβ‒Oγ bond is 1.467 Å in 1-EN2, it extends to 1.742 Å in 1-TS2. In 1-TS2, the geometry of catalytic phosphate is H3PO4-like. The 1TS2 has a single imaginary frequency of 569i cm−1. A proton from H2PO4− is passed to the Serresidue side-chain OH group, leading to the desorption of a water molecule from the enolized Ser residue. During the conversion of 1-EN2 to 1-PC, φ and ψ change by 34° and 20°, respectively. The 1-PC is composed by a Dha residue, one water molecule, and catalytic H2PO4−. The hydrogen bond between the Ser-residue main-chain NH proton and H2PO4− is retained throughout the Ser-residue dehydration. This hydrogen bond most likely plays an important role in fixing the catalyst at an appropriate position. The activation barrier for 1,3-elimination based on 1-RC was 32.7 kcal mol−1.

1-RC

1.946

1.725

1-EN1

1-TS1 1.813

2.104

2.722

1.864

1.481 1.954

2.347

1.701

2.306

1.167

2.466

1.084 1.376


1.953

1.630

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Figure 4. Calculated geometries of the enolization system: 1-RC (φ = 80°, ψ = −120°, χ1 = 79°, χ2 = −50°), 1-TS1 (φ = 77°, ψ = −139°, χ1 = 83°, χ2 = −44°), and 1-EN1 (φ = 92°, ψ = −153°, χ1 = 90°, χ2 = −44°). Selected interatomic distances are shown in Å.

1-EN2

1-TS2

1-PC

1.742 1.830 2.038

2.100 1.605

1.486 1.040

1.318 1.120

1.976

1.732

1.716

Figure 5. Calculated geometries of the 1,3-elimination system: 1-EN2 (φ = 88°, ψ = −163°, χ1 = −75°, χ2 = 172°), 1-TS2 (φ = 83°, ψ = −169°, χ1 = −76°, χ2 = −149°), and 1-PC (φ = 54°, ψ = −143°; χ1 and χ2 are not defined). Selected interatomic distances are shown in Å.

3.2.2. Pathway 2 Figure 6 shows the optimized geometries of the enolization system. During enolization, 2-RC is converted to 2-EN1 via 2-TS1. In 2-RC, the model compound has three interactions with H2PO4− including CαHα−O interaction and two hydrogen bonds; however, the mode of interaction in 2RC is different from that in 1-RC. The distance between CαHα−O interaction is 2.274 Å, which is shorter than that of 1-RC. During the conversion of 2-RC to 2-EN1, φ and ψ changed by 33° and 44°, respectively. In 2-TS1, the transfer of two protons progresses via H2PO4−. The single imaginary frequency of 2-TS1 is 1078i cm−1. Similar to Pathway 1, two hydrogen bonds formed between Ser-residue side chain and main chain are remarkably elongated. We speculate that side-



chain rotation will be easier because the Ser-residue side-chain restriction will weaken. The activation barrier of the enolization process was 21.2 kcal mol−1. 2-EN2 is formed by the rotation of the Ser-residue side chain of 2-EN1. Rotation of the Ser-residue side chain is a four-step process with insignificant changes. Figure S2 of the Supporting Information presents the energy minima and TS geometries found during the rotation of the Ser-residue side chain. The difference between the values of χ1 of 2-EN1 and 2-EN2 is 156°. In 2-EN2, a new hydrogen bond is formed between the Ser-residue side-chain OH group and catalytic H2PO4−. Figure 7 shows the optimized geometries of the 1,3-elimination system. In this process, 2-EN2 is converted to 2-PC via 2-TS2. When 2-EN2 is converted to 2-PC, cleavage of the Ser-residue side-chain Cβ−Oγ bond and double proton relay mediated by H2PO4− occurs. The distance of Cβ−Oγ is 1.472 Å in 2-EN2 but extends to 1.780 Å in 2-TS2. In 2-TS2, the geometry of catalytic phosphate is H3PO4-like. The single imaginary frequency of 2-TS2 is 531i cm−1. When 2-PC is formed, the proton was transferred to the Ser-residue side-chain OH group, and one water molecule is newly released. In the 1,3-elimination process, the catalytic H2PO4− rotates, and the Ser-residue main-chain NH proton forms hydrogen bonds with different oxygen atoms in H2PO4−. In addition, φ and ψ changed by 71° and 25°, respectively, and a large conformational change of the Ser-residue main chain was observed. The activation barrier for 1,3-elimination was 31.8 kcal mol−1, based on 2-RC.


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2-RC

2-EN1

2-TS1 1.722

1.954

2.104

1.813

1.864

1.481

2.170

2.274 1.719

1.167 2.306

2.722

2.466 1.084

1.953

1.376

1.630

Figure 6. Calculated geometries of the enolization system: 2-RC (φ = 86°, ψ = −121°, χ1 = 79°, χ2 = −54°), 2-TS1 (φ = 85°, ψ = −141°, χ1 = 84°, χ2 = −49°), and 2-EN1 (φ = 119°, ψ = −165°, χ1 = 89°, χ2 = −51°). Selected interatomic distances are shown in Å.

2-TS2

2-EN2

2-PC

1.780 1.852 1.934

1.649

1.449

1.339

2.243

1.111

1.976

1.732

1.053 1.716

Figure 7. Calculated geometries of the 1,3-elimination system: 2-EN2 (φ = 125°, ψ = −168°, χ1 = −67°, χ2 = 93°), 2-TS2 (φ = 107°, ψ = −167°, χ1 = −78°, χ2 = 92°), and 2-PC (φ = 54°, ψ = −143°; χ1 and χ2 are not defined). Selected interatomic distances are shown in Å.

3.2.3 Pathway 3 Figure 8 shows the optimized geometries of the enolization system. In this process, 3-RC is converted to 3-EN1 via 3-TS1. In 3-RC, the model compound forms three interactions with



H2PO4− including CαHα−O interaction and two hydrogen bonds; however, the mode of interactions in 3-RC is different from those in 1-RC and 2-RC. The distance of CαHα−O interaction is 2.323 Å. In 3-TS1, the geometry of catalytic phosphate is H3PO4-like, which is similar to that of 3-EN1 rather than 3-RC. During the conversion from 3-RC to 3-EN1, φ and ψ changed by 23° and 68°, respectively. In this pathway, the interaction between the N-terminal amide and side-chain OH group was weak (the distance of NH−Oγ is 2.449 Å), and a larger main-chain rotation occurred, as the enolization proceeded, compared with Pathways 1 and 2. The activation barrier of the enolization process was 23.0 kcal mol−1. 3-EN2 is formed by the rotation of the Ser-residue side chain of 3-EN1. The rotation of the Ser-residue side chain is a one-step process with insignificant changes. Figure S3 of the Supporting Information presents the energy minima and TS geometries found during the rotation of the Ser-residue side chain. The difference in χ1 between 3-EN1 and 3-EN2 is 174°. In 3-EN2, two hydrogen bonds are newly formed; one between the Ser-residue side-chain OH group and catalytic H2PO4−, and the other between Ser-residue side-chain OH group and amide C=O oxygen of N-terminal side adjacent residue. Figure 9 shows the optimized geometries of the 1,3-elimination system. In this process, 3-EN2 is converted to 3-PC via 3-TS2. Throughout the 1,3-elimination process, the hydrogen bond formed between Ser-residue side-chain OH proton and main-chain carbonyl oxygen of Cterminal side adjacent residue was retained. Since 1-EN2 and 2-EN2 do not possess hydrogen bond acceptors near the Ser-residue side chain, corresponding hydrogen bonds cannot be formed. When 3-EN2 is converted to 3-PC, cleavage of the Ser-residue side-chain Cβ−Oγ bond and the double proton relay mediated by H2PO4− occur. During the conversion of 3-EN2 to 3-TS2, the distance of Cβ−Oγ is extended from 1.459 Å to 1.620 Å. When 3-PC is formed, the Cβ−Oγ bond


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is cleaved, and one water molecule is newly formed. The activation barrier for 1,3-elimination was 30.4 kcal mol−1, based on 3-RC. In the 1,3-elimination process, φ and ψ changed by 10° and 14°, respectively. This change in φ and ψ is the smallest among the three pathways investigated in this study. This small conformational change in the main chain may contribute to a reduction in the activation barrier.

3-RC

3-EN1

3-TS1 2.004

2.323

2.340

2.287

2.449

1.907

1.760

1.784

1.116

1.692

1.319 1.018

1.767

1.537

Figure 8. Calculated geometries of the enolization system: 3-RC (φ = −104°, ψ = −86°, χ1 = 56°, χ2 = −178°), 3-TS1 (φ = −134°, ψ = −147°, χ1 = 81°, χ2 = −179°), and 3-EN1 (φ = −127°, ψ = −154°, χ1 = 91°, χ2 = 179°). Selected interatomic distances are shown in Å.

3-EN2

3-PC

3-TS2

1.620 1.813 1.817

1.547

1.957

1.647 1.260

1.696 1.798

1.165

1.007


2.083 1.905

1.797 1.754


Figure 9. Calculated geometries of the 1,3-elimination system; 3-EN2 (φ = −129°, ψ = −158°, χ1 = −83°, χ2 = 62°), 3-TS2 (φ = −120°, ψ = −166°, χ1 = −78°, χ2 = −60°), and 3-PC (φ = −119°, ψ = −144°. χ1 and χ2 are not defined). Selected interatomic distances are shown in Å.

3.2.4. Energy profile Figure 10 shows the energy profile of Dha-residue formation via three pathways. In the Supporting Information, Tables S1‒S8 present the total MP2 and B3LYP energies, ZPEs, hydration free energies of all energy minima and TS geometries. In addition, Tables S9‒S44 present the Cartesian coordinates of all energy minima and TS geometries. In all the enolization processes of Pathways 1, 2, and 3, the catalytic H2PO4− was placed on the side to which the αproton was originally bound, so that the catalytic H2PO4− abstracts the α-proton of the Ser residue. In reaction pathways 1, 2, and 3, the local activation barriers in the enolization process were 23.3 kcal mol−1, 21.2 kcal mol−1, and 23.0 kcal mol−1, respectively. Since 2-RC is more unstable than 1-RC, the energy of 2-TS1 is higher than that of 1-TS1 by 0.596 kcal mol−1. On the other hand, although the conformations of 1-RC and 3-RC are largely different, 1-RC is as stable as 3-RC, and there is no significant difference in the activation barriers of 1-RC and 3-RC in the enolization process. This suggests that the conformation of Ser residues, which are prone to enolization, is not unique. There is no significant difference in the energy of all enolization products including 1-EN1, 2-EN1, and 3-EN1. Enolized Ser residues also act as intermediates in the Ser-residue stereoinversion. When the Ser residue undergoes enolization, Cα converts from sp3- to sp2-hybridized states, and the asymmetric carbon vanishes. Ser is prone to stereoinversion in peptides and proteins, in addition to Asp. Fujii et al. reported that the activation barrier for Asp-residue stereoinversion is 21.4‒28.3 kcal mol−1 in phosphate buffer45. Surprisingly, the


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calculated activation barrier of Ser-residue stereoinversion was reproduced this value. However, the ease of stereoinversion of amino acid residues is not determined only by the activation energy. If the amino acid residue is not exposed on the protein surface, catalytic molecules are not properly placed around the amino acid residue, and the conformational change of the amino acid residue is restricted. Thus, the stereoinversion of Ser residue is more difficult than that of Asp residue, probably because Ser is less polar than Asp and is less likely to be exposed on the protein surface. If the Ser residue undergoes enolization, and 1,3-elimination of the enolized Ser residue does not proceed, D-Ser residue is formed by the ketonization of enolized Ser. That is, Ser-residue dehydration can compete the stereoinversion.

1,3-elimination

Relative energy / kcal mol−1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

Enolization

Side-chain rotation

Pathway 1 Pathway 2 Pathway 3

20 10 0

Reaction pathway

Figure 10. Energy profile of Dha-residue formation via Pathway 1, Pathway 2, and Pathway 3. All the relative energies were calculated at the MP2/6-311+G(d,p) level of theory and were corrected for ZPEs.

It is necessary that the side chain of Ser residue undergoes remarkable rotation for the products of Ser-residue enolization (1-EN1, 2-EN1, and 3-EN1) to function as reactants in the



1,3-elimination process (1-EN2, 2-EN2, and 3-EN2) without significant catalytic-H2PO4− migration, since catalytic H2PO4− needs to catalyze the proton relay between the enol and sidechain OH groups. In 1-EN1 and 2-EN1, the Ser-residue side-chain OH group interacts with the main-chain of adjacent residues, and it is considered that these interactions hinder the rotation of Ser-residue side chain. However, in the enolization process, Cα changes from sp3- to sp2hybridized state, and these interactions are weakened. This suggests that the restricted rotation of the Ser-residue side chain is relaxed, thus allowing the rotation to occur. The products of 1,3elimination, 1-PC and 2-PC, are highly similar, and 1-PC is considered to be converted to more stable 2-PC. The calculated energies of all 1,3-elimination products (1-PC, 2-PC, and 3-PC) were slightly lower than those of the reactants (1-RC, 2-RC, and 3-RC), and Dha and Ser residues are predicted to have comparable stability. The high stability of Dha residues derived by quantum chemical calculation supports the experimental result that Dha residues are very stable in the absence of nucleophiles24. In the 1,3-elimination process, the relative energy of 1-TS2, 2-TS2, and 3-TS2 with respect to 3-RC, which is the most stable reactant, is estimated as 32.7 kcal mol−1, 34.4 kcal mol−1, and 30.4 kcal mol−1, respectively. The local activation energies of the 1,3-elimination process, which is considered a rate-determining step, were 11.1 kcal mol−1 and 10.1 kcal mol−1 in Pathways 1 and 3, respectively. Since the difference in the activation barriers of Pathways 1 and 3 is not high enough to determine which pathway is more favorable, it is considered that Ser-residue dehydration can proceed in both pathways. In the gas phase, the relative energies of 1-TS2, 2-TS2, and 3-TS2 with respect to 1-RC, which is the most stable reactant in the gas phase, were estimated as 33.2 kcal mol−1, 31.8 kcal mol−1, and 31.9 kcal mol−1, respectively; there were no substantial differences between the activation barriers in the gas phase and those in the aqueous phase. Therefore, it appears that the hydration free energies


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do not greatly affect the Ser-residue dehydration mechanism. On the other hand, during the whole 1,3-elimination process, the catalytic H2PO4− was arranged on the side corresponding to the face to which the α-proton was originally bound. However, it is also conceivable that the catalytic H2PO4− is placed on the opposite side and catalyzes the 1,3-elimination. Note that in this case the Ser-residue side chain does not need to rotate, as the formation of hydrogen bonds between the Ser-residue enol and side-chain OH groups, and H2PO4− is necessary. In this case, both 1,3-elimination pathways are mirror images of the abovementioned process, and the activation barriers of these pathways are considered to be equal. Although this suggests that the rotation of the Ser-residue side-chain rotation is not essential for the Ser-residue dehydration process if other reactions do not occur, in this case, it is considered that ketonization with lower activation energies (2.52 kcal mol−1, 4.12 kcal mol−1, and 2.02 kcal mol−1 in Pathways 1, 2, and 3, respectively) proceeds preferentially to 1,3-elimination (with formation of the D-Ser residue). Since the activation barrier of 1,3-elimination process is markedly higher than that of the enolization process in all three pathways investigated in this study, we suggest that the ratedetermining step of nonenzymatic Ser-residue dehydration is 1,3-elimination.

4.

CONCLUSIONS

In the present study, three-type complexes, comprising the model compound Ace-Ser-Nme and H2PO4−, were used as reactants, and the plausible Dha-residue formation pathway was obtained by quantum chemical calculations. Two types of conformers were used (i.e., R1 and R2), and the conformer R1 interacted with catalytic H2PO4− in two ways, whereas R2 interacted with one position of H2PO4−. Although 3-RC is formed by placing H2PO4− around the optimized model compound R2, 3-RC is comparatively more unstable than 1-RC and 2-RC in the gas phase, and



3-RC is predicted to be stabilized by hydration. Thus, it is conceivable that 3-RC conformation can easily exist in the aqueous phase. The Dha-residue formation consists of two steps: enolization and 1,3-elimination. To date no studies have reported that Dha-residue formation can proceed enzymatically. Therefore, the obtained activation energies for Ser-residue dehydration obtained in the present study compared with that of nonenzymatic Asp-residue stereoinversion, which was confirmed to occur experimentally. The calculated activation barrier for the Serresidue dehydration were comparable to that obtained for Asp-residue stereoinversion, which is known to proceed in vivo45. However, nonenzymatic Ser-residue dehydration may compete with stereoinversion, which has a lower activation barrier, while the regeneration of Ser residue from enolized Ser is almost barrier-less. For this reason, it is considered that the regeneration of L-Ser residue from enolized Ser residue has priority over the dehydration and stereoinversion. On the other hand, to continue the Ser-residue stereoinversion, it is necessary to arrange the catalyst molecules on the opposite sides of the face to which the α-proton was originally bound. Therefore, Ser residue can undergo dehydration in environments without this requirement for protonation. In the present study, the simplified model compound (Ace-Ser-Nme) was used and no consideration was obtained regarding the influence of the protein’s three-dimensional structure on the Ser-residue dehydration. Since Ser-residue dehydration mechanism has not been previously proposed or investigated, we hypothesized for the first time the Ser-residue dehydration mechanism using a simplified model compound. Further studies using more complex models are needed to clarify the influence of the protein environment on the Ser-residue dehydration. Since the catalytic H2PO4− may interact with model molecule in different ways when large model molecules are used, comprehensive studies using molecular dynamic


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simulations should be conducted. We are currently planning such studies. On the other hand, phosphate species do not always exist around the protein. Since, under physiological conditions, water always exists around the protein, it is conceivable that the water molecules catalyze the Ser-residue dehydration. However, to date we did not succeed in finding a water-catalyzed Serresidue dehydration pathway with low activation barrier. We will report the water-catalyzed Serresidue dehydration pathways in a future study.

ACKNOWLEDGMENTS This work was supported by grants-in-aid for scientific research [17K08257] from the Japan Society for the Promotion of Science.

Supporting Information Figure showing calculated geometries of the Ser-residue side-chain rotation system, tables showing total energies, zero-point energies, and hydration free energies of all the optimized geometries, and tables showing Cartesian coordinates of all the optimized geometries.

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Table of Contents Image −1

Dehydration (rate determining)

Relative energy / kcal mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 20 10 0

Pathway 1 Pathway 2 Pathway 3

Side-chain rotation Enolization Ser (keto form) O N H H

OH H N O

Dehydroalanine O N H

OH H N

O N H

H N O

OH

Ser (enol tautomer)

Reaction pathway


29

Possible Mechanisms of Nonenzymatic Formation of Dehydroalanine

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