- Email: [email protected]
Effect of organic molecules on hydrolysis of peptide bond: A DFT study
Chemical Physics 415 (2013) 282–290
Contents lists available at SciVerse ScienceDirect
Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Effect of organic molecules on hydrolysis of peptide bond: A DFT study Olga Makshakova, Elena Ermakova ⇑ Kazan Institute of Biochemistry and Biophysics RAS, 420111 Kazan, P.O. Box 30, Russia
a r t i c l e
i n f o
Article history: Received 2 November 2012 In final form 30 January 2013 Available online 9 February 2013 Keywords: Density functional theory Transition state Dialanine Organic solvent
a b s t r a c t The activation and inhibition effects of small organic molecules on peptide hydrolysis have been studied using a model compound dialanine and DFT approach. Solvent-assisted and non-assisted concerted mechanisms were analyzed. Several transition states for the systems: alanine dipeptide–water molecule in complexes with alcohol molecules, acetonitrile, dimethylsulfoxide, propionic, lactic and pyruvic acids and water molecules were localized. The formation of hydrogen bonds between dipeptide, reactive water molecule and molecules of solvents influences the activation energy barrier of the peptide bond hydrolytic reaction. Strong effect of organic acids on the activation energy barrier correlates with their electronegativity. Acetonitrile can act as an inhibitor of reaction. Mechanisms of regulation of the activation energy barrier are discussed in the terms of donor-acceptor interactions. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The peptide bond cleavage is a major process of protein decomposition and metabolism of living matter. The covalent stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Both enzymatic and nonenzymatic hydrolysis of peptide bond is of biological and technological significance [1–4]. The reaction mechanism of nonenzymatic peptide hydrolysis has been studied from many points of view [5–19] and is still an object of theoretical investigation by modern calculation methods [10–18]. From the theoretical point of view, the peptide bond has been modeled using different compounds like formamide [10– 15], N-methylacetamide [16–18], N,N-dimethylformamide [19] and the mechanisms of bond cleavage have been treated at different levels of theory, including cluster analysis [10–14] and ab initio CPMD simulations [16–18]. At present four different mechanisms of hydrolysis are discussed: the concerted mechanism with and without the additional water molecule and the stepwise mechanism with and without the additional water molecule. In the concerted mechanism, the addition of the water molecule and cleavage of the CN amide bond occur in a single step [13]. In the stepwise mechanism, water is first added to the CO bond to form a gem-diol intermediate and subsequently the proton transfer from one of the OH groups to the nitrogen atom induces the dissociation of the system and formation of the products. Participation of the second water molecule in each
⇑ Corresponding author. Tel.: +7 843 2319037; fax: +7 843 2927347. E-mail address: [email protected] (E. Ermakova). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.01.037
mechanism (the water-assisted mechanism) decreases the activation energy. According to the findings of Refs. [11–13], nucleophilic attack on carbon atom is the rate limiting step of all reaction mechanism and the energy barriers for the concerted mechanism and the stepwise one are nearly close (1 kcal/mol). CPMD calculations found no intermediate in the reaction dynamics and showed that the concerted attack of carbon and nitrogen atoms by water molecules directly results to products [17]. We believe that the effect of organic solvent molecules at the first stage of the stepwise pathway is qualitatively similar to their effects on the concerted mechanism studied here. The detailed analysis of the stepwise mechanism is out of scope of the present work. Here we only deal with the concerted mechanism. It is well known that the physicochemical properties of environment have crucial impact on the rates of both chemical and biochemical reactions. Different theoretical approaches have been developed over the years to account for water as an essential living environment. Water environment was taken into account both explicitly [13,16–18] or implicitly by continuum models [11,20] or by means of the combined model of solvent. In the combined model the central compound was solvated explicitly by some solvent molecules and the resulting cluster was treated by a dielectric continuum model [14]. Live cells are crowded with high- and low-molecular organic compounds which change the thermodynamic activity of water and can specifically interact with reagents, transition states, intermediates and products of reactions [5]. Such organic molecules as alcohols and acids can regulate the rate of the hydrolytic reaction indirectly as well as interact with peptide inducing a peptide bond breaking. The direct interaction is intensively studied experimentally and theoretically [19].
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Main aim of our investigation was to define an indirect influence of the organic molecules on the peptide bond hydrolysis thorough their interaction with reactive water molecule. In this study we have focused attention on the influence of water molecule and organic solvent molecules on the concerted mechanism of hydrolysis, on the structure and energy of transition state and on the activation energy for nonenzymatic hydrolytic reaction using a model compound, dialanine, and DFT computational method. Several organic solvents with significant technological importance were considered: methanol, ethanol, 1-propanol, acetonitrile (AN), dimethylsulfoxide (DMSO), carboxylic acids (propionic, lactic and pyruvic). To our knowledge, there have not been previously reported systematic investigations of the structural and energetic aspects of the peptide bond hydrolysis in the presence of organic solvent molecules in literature. Understanding of the influence of organic compounds on nonenzymatic hydrolysis reactions can provide a new insight into mechanism of non-aqueous enzymatic reactions where water–organic solvent is used as reaction media [4]. The information is also can be useful for the development of novel drugs based on transition state or intermediate structures. 2. Theoretical approach The initial structure of alanine dipeptide (hereafter dipeptide) was slightly modified by substituting of hydroxyl fragment of carboxyl group of C-terminus and of a hydrogen atom of N-terminus with methyl groups in order to weaken their influence on the studied reaction. The DFT method using hybrid functional B3LYP [21,22] was applied for the location of minima and transition states (TS) structures. The Pople’s split-valence basis set 6-31G [23] was used including the polarization and diffusion functions 6-31G(d), 6-31+G(d, p), 6-31+G(2df, p), 6-31++G(2df, p), and 6311++G(2df, 2p). Structural parameters and energies for reactants and transition states were defined using the program package Gaussian09 [24]. The location of TS structures was performed starting from the dipeptide initial structure and adding a water molecule to the peptide bond. The geometries of all transition states and reactants were fully optimized at corresponding level of theory. The transition state structures were verified by the analysis of the normal vibration frequencies. The correlation between stable structures and transition states was checked by analysis of the corresponding imaginary frequency mode and by the intrinsic reaction coordinate (IRC) calculations. The stationary structures were recognized as true minima after verifying the lack of imaginary frequencies. The activation energy was calculated using the following formula:
E ¼ ETS Epept EW
283
lactic and pyruvic acids were used to study the influence of the explicit solvent on the activation energy barrier. We repeated the search for transition states including one low-weight organic molecule or one extra water molecule. Calculations were performed in gas phase conditions using B3LYP/6-31G(d) basis set. The activation energy was calculated from the formula:
E ¼ ETS Epept EW ES
ð2Þ
where Es is energy of explicit solvent and other designations are the same as in (1). 3. Results and discussion 3.1. Non-assisted mechanism First we have studied the well known concerted non-assisted mechanism of peptide bond hydrolysis to compare our calculations with the results obtained earlier [11,13]. According to this mechanism, single water molecule forms with dipeptide the four-member ring, which was previously described by Antonczak et al. [11] for formamide hydrolysis. Fig. 1 depicts the geometry of the transition state (TS). Table 1S of Supplementary contains some geometry parameters of the transition state. The predicted TS geometry of dialanine is very similar to the geometry of transition state of formamide hydrolysis obtained at the MP2 level with 6311++G(d, p) basis set [15]. The comparison of the activation energies calculated using different basis sets (Table 1) evidences a strong decrease of the energy values of reagents with the increase of the basis set from 6-31G(d) to 6-311++G(2df, 2p). At the same time, the energy values of the transition state are slowly decreased with the basis set augmentation. As a result, one can see the increase of the activation energy barrier of the hydrolytic reaction along with the basis set augmentation. Gorb et al. [13] applied different methods and basis sets for calculation of transition state in the reaction of formamide hydrolysis in water. The comparison of both the activation energies and free energy barriers obtained with B3LYP/6-31G(d) and MP2/6311+G(d, p) revealed a good qualitative agreement between both methods but the B3LYP provided smaller values than MP2. The comparison of the potential energies calculated at different levels and different basis sets (B3LYP/6-31+G(d, p), B3LYP/cc-pVTZ, MP2/cc-pVTZ) was also carried out in Ref. [18]. DE was found in the range from 40.4 to 43.3 kcal/mol depending on the basis set and method.
ð1Þ
where, ETS, Epept and EW are the energy of transition state, dipeptide and water molecule. All energy values were corrected taking into account the zero-point vibrational energies. For TS, zero-point vibrational energy was calculated missing the imaginary frequency. The detailed analysis of implicit influence of bulk solvent was performed for non-assisted mechanism. Four different continuum models were used (polarizable continuum mode (PCM) [25,26], polarizable conductor calculation model (CPCM) [27], isodensity polarizable continuum mode (IPCM) [28], and solvation model of density (SMD) [29] to take into account the effects of bulk water. Only PCM model was used for organic solvents. Dielectric permeability were 78.36, 24.85, 35.69, and 46.83 for water, ethanol, AN, and DMSO, respectively. Molecules of organic solvents: methanol, ethanol, 1-propanol, acetonitrile, DMSO, and molecules of carboxylic acids: propionic,
Fig. 1. Transition state for the gas phase reaction of the peptide hydrolysis in nonassisted mechanism.
284
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Table 1 Energies of the transition and ground states (Hartrees) and the activation energies (kcal/mol) of the peptide bond hydrolysis calculated with B3LYP functional at different basis sets.
ETS Epept Ew DE TDS DG
6-31G(d)
6-31+G(d, p)
6-31+G(2df ,p)
6-31++G(2df, p)
6-311++G(2df, 2p)
650.7902794 574.444677 76.4089533 40 12.5 51
650.8578779 574.4950466 76.4340477 44.8 12.7 56
650.8858427 574.5187152 76.4406146 46.2 12.6 57.4
650.886186 574.5189777 76.4406664 46.2 12.6 57.4
651.0423138 574.6549343 76.4626211 47.2 12.4 58.2
DE = ETS Epept Ew. TDS = T(STS Spept SW). DG = GTS Gpept GW. Subscripts designate: w –water molecule, pept – dipeptide.
It is well known that entropic effects play a crucial role in the reactivity of weakly bonded complexes. Large negative TDS values were obtained for the water assisted and non-assisted formamide hydrolysis with amount 10–12 kcal/mol per water molecule [12]. However, it was demonstrated that entropy terms evaluated from the theoretical computations referring to gas phase processes were significantly overestimated [30,31]. Consequently the calculated free energies were overestimated as well. Different scientific groups reported free energy values ranging from 35 to 60 kcal/mol [11,13,32]. Estiu et al. [32] have showed that the primary origin of the difference between the values reported by different groups was based on the definition of the initial state. The calculation of the free energy barrier relative to the separated reagents (see Theoretical approach) with 6-311++G(2df, 2p) basis set gives 58 kcal/mol (Table 1), that is very close to results of Estiu et al. [32] obtained with MP2/6-311++G⁄⁄ level of theory. Taking into account the solvent effect as a continuum producing the polarization of solute we can evaluate the media effect on molecular complexes. We compared several continuum solvent models to estimate the role of environmental water in stabilization of the transition state of the dipeptide hydrolysis. Four different continuum models demonstrate the similar results (Table 2S). The comparison of main geometry parameters of TS reveals a small increase of the length of OH breaking bond and a decrease of the CN peptide bond for three continuum models (PCM, CPCM, and SMD). The distance between the O atom of water molecule and the C atom of peptide slightly elongates and the distance between the H atom of water and the N atom of peptide shortens (Table 2S). The implicit account of environmental water reduces the energy of both the reagents and the TS. Taking account of bulk water by PCM decreases the transition state energy by about 10 kcal/mol. At the same time the energies of the dipeptide and water molecule are reduced by 7.5 and 4.5 kcal/mol, respectively, compared with the results in the gas phase. Thereby the activation energy growth by about 2 kcal/mol is observed. SMD gives the lowest values of the energy of both the reagents and the TS. SMD stabilizes TS, peptide and water molecule by 19, 12, and 8.2 kcal/mol, respectively, compared with the results in the gas phase. Implicitly treated solvents demonstrated rather small effect in Ref. [13], the largest change in DG was less than 2 kcal/mol. This is in agreement with the previous suggestion made by Antonczak et al. [11]. Similar slight effect of continuum solvent was found in the recent study of enzymatic peptide hydrolysis [33]. Unfortunately PCM demonstrates no effect of solution dielectric constant on the activation energy. The activation energy for all used organic solvents, 41.7 kcal/mol, is very similar to the result found for bulk water (Table 3S). Comparing the results obtained with different basis sets and different dielectric continuum solvent models we conclude that implicitly treated solvents demonstrate rather small effect on the activation energy and B3LYP method with small basis set
6-31G(d) slightly underestimates the activation energy barrier, but it gives a better agreement with the experimental data [7,8]. Additionally, for large biomolecule or for ab initio MD studies 631G(d) basis set is considered to be the best compromise of speed and accuracy, and is the most commonly used [33]. 3.2. Influence of organic molecule on the transition state energy in non-assisted mechanism Here we focus attention on one point of hydrolysis in a complicated system of peptide, water and organic compounds where the organic molecules influence the water molecule – peptide interactions by means of hydrogen bond formation with the reactive water molecule. The hydrogen bonding complexes of two types were taken into account. In complexes of the first type, an organic molecule acts as a lone pair electron acceptor and interacts with the oxygen atom of water molecule. In complexes of the second type, an organic molecule acts as a lone pair electron donor and interacts with a hydrogen atom of water molecule. Keeping in mind that the entropy contributions to free energy barriers are strongly overestimated compared to the experiment [30,31] we will further discuss the influence of solvent molecule on the activation energy. Calculated values of free energy are also presented in the tables. The first structure (TS_w_C1) possesses an additional water molecule bound to the O atom of reactive water molecule (Fig. 2, Table 4S). The energetic characteristics of the transition state are listed in Table 2. The activation energy barrier for this complex is 32.8 kcal/mol. Since we calculate the energy barrier relative to the separated reactive molecules, the formation of hydrogen bond between two water molecules makes difficult direct comparison of the energy barriers of TS_w_C1 and TS. In the second structure (TS_w_C2) an additional water molecule forms two hydrogen bonds – the O atom is bound to the H atom of reactive water molecule and the H atom of the additional water molecule is bound to the carboxyl O atom of peptide group (Fig. 2). In this ring-like structure the network of several covalent and hydrogen bonds forms a closed structure. Two hydrogen bonds (TS_w_C2 complex) lower the activation energy down to 29.6 kcal/ mol. The influence of methanol, ethanol and propanol molecules on the TS of hydrolysis reaction was investigated (Table 4S). The interaction of alcohol molecule with peptide and water molecule slightly changes the geometry parameters of TS and the activation energy. Two types of different complexes were found for all alcohols. For the first type of complexes (TS_MeOH_C1, TS_EtOH_C1, TS_PrOH_C1), the hydrogen bond formed between the oxygen atom of water molecule and the hydrogen atom of the hydroxyl group of alcohol molecules decreases the energy of the transition state. Interactions of the type of C–H. . .O and C–H...N give an
285
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Fig. 2. Transition states for non-assisted mechanism with additional water molecule.
Table 2 Activation energies (kcal/mol) of the peptide bond hydrolysis in the complexes with water molecule and alcohol molecules in non-assisted concerted mechanism.
DE TDS DG
w_C1
w_C2
MeOH_C1
MeOH_C2
EtOH_C1
EtOH_C2
PrOH_C1
PrOH_C2
32.8 21.6 52.4
29.6 23.1 50.3
30.7 22.5 51.7
27.5 23.4 49.1
30.6 22.4 51.7
27.7 23.6 49.6
30.5 22.6 51.8
27.6 23.3 49.3
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
additional stabilization of complex TS. The importance of weak hydrogen bonds of the type of C–H. . .O and C–H...N is detailed discussed elsewhere [34]. The formation of these complexes leads to very similar activation energies – 30.7, 30.6, 30.5 kcal/mol for methanol, ethanol, and propanol, respectively (Fig. 3, Table 2). The second type of the transition states in complexes with alcohol molecules (TS_MeOH_C2, TS_EtOH_C2, TS_PrOH_C2) is stabilized by two hydrogen bonds forming ring-like structure, respectively. The activation energy of these complexes drops down
to 27.5, 27.7, and 27.6 kcal/mol, for methanol, ethanol, and propanol, respectively. Structures of the complexes of dipeptide with alcohol molecules are similar to the structures of appropriate complexes with water molecule TS_w_C1 and TS_w_C2. The activation energies of dipeptide – water – alcohol systems are similar to each other and are slightly lower than values in dipeptide – water – water systems. Aprotic organic solvent molecules – acetonitrile (AN) and dimethylsulfoxide (DMSO) – have a different influence on the TS
Fig. 3. Transition states for non-assisted mechanism with additional methanol, ethanol and 1-propanol molecules.
286
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
of hydrolysis. Two different transition states for the system of AN – peptide – water were found where AN molecule interacted with water molecule and with peptide by both methyl group and the N atom. In the first complex (TS_AN_C1), AN molecule interacts with water by the methyl group (Fig. 4, Table 5S) and forms two weak bonds between the H atoms of AN and the O atom of water molecule and the N atom of N-terminus of dipeptide. The activation energy barrier for the first type of hydrolysis reaction is higher and is equal to 35.6 kcal/mol (Fig. 4, Table 3). In the second complex (TS_AN_C2), acetonitrile molecule interacts with water molecule by the nitrogen atom and forms the N. . .HOH hydrogen bond between the N atom of AN and the hydrogen atom of water. The second weak hydrogen bond of type CH. . .O is formed between the hydrogen atom of the methyl group of AN and the carbonyl O atom of dipeptide. The activation energy barrier is lower than one of TS_AN_C1 and equals 33.6 kcal/mol (Table 3). One can see that AN weakly interacts with reactants and demonstrates a higher activation energy barrier of the reaction. Dimethylsulfoxide has a strong influence on the peptide hydrolysis as well. Two transition states for the system of DMSO – peptide – water were found where molecule of DMSO interacted with water molecule by the O atom and by the S atom (Fig. 5, Table 5S). In the first TS_DMSO_C1 complex the S atom of DMSO is bound to the H atom of the reactive water molecule, the O atom is bound to the H atom of methyl group of the peptide, and weak hydrogen bond of type CH. . .O is formed between the H atom of the methyl group of DMSO and the O atom of the peptide. This complex is less energetically favorable than TS_DMSO_C2. The formation of this complex results in the activation energy of 31.5 kcal/mol (Table 3). In more favorable TS_DMSO_C2 transition state, one hydrogen bond between the O atom of DMSO and the H atom of water is formed. Two weak bonds between the H atoms of the methyl groups of DMSO and the O atom of peptide group and the N atom of N-terminus of the peptide give additional stabilization for this TS. The formation of this complex leads to the lowering of the activation energy down to 26.3 kcal/mol. 3.3. Effect of carboxylic acid molecules on the transition state energy Three weak acids with different electronegativity were chosen to study the influence of carboxylic acid on the peptide bond hydrolysis. Propionic acid (PrA) is a carboxylic acid with chemical formula CH3CH2COOH. Lactic acid (LA) with the chemical formula CH3CHOHCOOH has a hydroxyl group adjacent to the carboxyl group. Pyruvic acid (PA) (CH3COCOOH) contains oxygen atom at the second carbon position. Electronegative substituents make acids stronger. Electronegativity of these acids increases from propionic acid to lactic and to pyruvic acid. Carboxylic acids are typically weak acids, implying that they only partially dissociate into
Table 3 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with AN and DMSO molecules in non-assisted concerted mechanism.
DE TDS DG
AN_C1
AN_C2
DMSO_C1
DMSO_C2
35.6 20.5 55.2
33.6 21.7 54.2
31.5 23.3 53.7
26.3 23.9 48.8
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
H + cations and RCOO–anions in neutral aqueous solution. We have taken into account only nonionized states of carboxylic acid molecules. Structures of complexes with lactic acid are given in Fig. 6. Geometric parameters and energy of the complexes are given in Table 6S and Table 4. For carboxylic acid molecules in nonionized state, we treated two types of complexes similar to those with water, alcohol, and aprotic organic solvent molecules. Thus, in the complexes of the first type (TS_PrA_C1, TS_PA_C1, TS_LA_C1), the oxygen atom of water molecule forms a strong hydrogen bond with hydroxyl group of carboxyl fragment of acid molecule. The second slight hydrogen bond is formed between the hydrogen atom of water and the carbonyl oxygen atom of acid molecule. Activation energies are equal to 26.9, 24.8 and 24.8 kcal/ mol for propionic, lactic and pyruvic acids, respectively. In the complexes of the second type (TS_PrA_C2, TS_PA_C2, TS_LA_C2), the hydrogen atom of water molecule creates hydrogen bound to the carbonyl oxygen of carboxylic acid molecule. Additional hydrogen bond is formed between the hydrogen atom of carboxyl group of the acid molecule and the carbonyl oxygen of the dipeptide. This type of the complexes is more favorable than the previous one; the energies are 23.2, 23 and 23.1 kcal/mol for PrA, LA and PA, respectively. 3.4. Solvent molecule-assisted mechanism If two water molecules participate in the bond cleavage, such reaction is referred to as water-assisted pathway. In this reaction the nucleophilic attack of one water molecule (W1) on the carbonyl carbon of peptide is accompanied by the concerted proton transfer of the second water molecule (W2) to the nitrogen atom of peptide (Fig. 7). The geometry of TS2_w_C1 is very similar to geometry of the transition state of water-assisted hydrolysis of formamide molecule obtained by Tolosa et al. with 6-311++G⁄⁄ basis set [15]. In the transition state TS2_w_C1 of water-assisted process, a hydrogen atom of the first water molecule (that performs nucleophylic attack) is situated between two oxygen atoms belonging to W1 water molecule and W2 molecule, the distances
Fig. 4. Transition states for non-assisted mechanism with additional acetonitrile molecule.
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
287
Fig. 5. Transition states for non-assisted mechanism with DMSO molecule.
Fig. 6. Transition states for non-assisted mechanism with lactic acid molecule in neutral form.
Table 4 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with carboxyl acid molecules in non-assisted concerted mechanism.
DE TDS DG
PrA_C1
PrA_C2
LA_C1
LA_C2
PA_C1
PA_C2
26.9 21.8 47.6
23.2 24.2 45.9
24.8 21.9 45.6
23 24.3 44.8
24.8 21.8 45.5
23.1 23.9 45.5
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule
H. . .O are 1.203 and 1.219 Å, respectively (Table 7S). The OH bond of W2 water molecule is elongated up to 1.347 Å. Transition state of water-assisted hydrolysis TS2_w_C1 is accompanied by lowering the activation energy down to 25.9 kcal/mol (Table 5). This value is in good agreement with previous results for water-assisted hydrolysis of formamide. The activation energy of formamide hydrolysis was found to reduce by 9 kcal/mol for the assisted concerted mechanism [13]. Further, one can note that the activation energy of TS2_w_C1 complex is lower than non-assisted reaction with the additional water molecule TS_w_C1 and TS_w_C2. In solvent-assisted mechanism, the nucleophilic attack is similar to water-assisted concerted mechanism; solvent molecule plays a role of the second water molecule. Transfer of the proton to the nitrogen atom of dipeptide occurs with the aid of catalytic solvent molecule. Solvent molecule accepts the hydrogen atom from water
molecule and donates its own proton to the nitrogen atom of peptide. Transition states corresponding to alcohol-assisted hydrolytic process are more energetically favorable than ones involved in water-assisted pathway by 4–5 kcal/mol. Hydrogen atom abstracted from water molecule is shifted to the proton acceptor atom of catalytic alcohol molecule; at the same time the OH bond of alcohol molecule elongates up to 1.31–1.32 Å. Three investigated alcohols give similar TSs and the activation energy barriers (Table 7S, Fig. 7). It is known that acetonitrile molecule is able to donate proton of its methyl group because of the strong electronegativity of C„N group. The transition state structure TS2_AN is given in Fig. 7. Methyl group of AN accepts a proton of water molecule and donates one to the N atom of peptide bond. The methyl C–H bond strongly elongates from 1.094 up to 2.264 Å. In AN-assisted mechanism the activation energy barrier significantly increases up to 51.8 kcal/mol. In other words, AN acts as an inhibitor of reaction. The studied carboxylic acids acting as catalysts lower the activation energy down to 14–16 kcal/mol. The hydrogen atom abstracted from water molecule is shifted to the oxygen atom of acid molecule and the OH bond of acid molecule elongates up to 1.57–1.64 Å. The activation energies increase in the range of lactic, pyruvic and propionic acids and are equal to 14.0, 14.2 and 16 kcal/ mol, respectively. (Table 7S, Fig. 8). The action of carboxylic acid in the concerted mechanism is similar to the hydrolysis mechanism of aspartyl proteases which was studied in Ref. [33] and refs therein. The activation energy obtained relative to pre-reaction complex was 22 kcal/mol and
288
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Fig. 7. Transition states for solvent-assisted mechanism with water molecule, methanol, and acetonitrile.
Table 5 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with water and organic molecules in solvent-assisted concerted mechanism.
DE TDS DG
w
MeOH
EtOH
PrOH
AN
PrA
LA
PA
25.9 23.85 46.8
21.3 23.8 43.2
21.3 24.3 43.8
21 23.9 43.3
51.8 24.8 74.8
16 24.8 39.2
14 25.2 37.6
14.2 24.6 37.3
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
should be compared with the free energy barriers obtained here which were in the range from 35 to 39 kcal/mol. It is not surprising that organic molecules act less effectively than proteins. 3.5. Comparison of the influence of organic compounds on the activation energies and on geometry parameters of the transition state In non-assisted mechanism of the peptide hydrolysis, organic compounds demonstrate a different effect on the activation energy of the reaction. Comparing with the additional water molecule contribution, organic molecules can decrease or increase the activation energy of the reaction. Molecules of three studied alcohols decrease the activation energy by 2 kcal/mol, carboxyl acids decrease it by 6–8 kcal/mol, molecule of AN increases the barrier by 4 kcal/mol. Molecule of DMSO decreases the activation energy by 1 kcal/mol. The changes of the activation energy barrier are accompanied by the modifications of geometry parameters of transition state. The discussed complexes can be divided into two groups. The first group includes the complexes where solvent molecule interacts by its H atom with the O atom of reactive water molecule or in other words it acts as the acceptor of the lone pair electrons. The complexes labeled above as C1 (except TS_DMSO_C1) belong to the
first group. The second group contains the complexes where the solvent molecule interplays by its O atom with the H atom of reactive water molecule and donates the lone pair electrons. The complexes of these two groups influence the geometry parameters of the transition state contrariwise. The length of the OH breaking bond of water molecule elongates along with the lowering of the activation energy in the first group of complexes. This finding is in agreement with the theory of the donor-acceptor interactions. The oxygen atom of water molecule donates the lone pair of electrons to solvent molecule. The electron density of the OH bond shifts to the water oxygen atom and induces the elongation of breaking OH bond. The formation of the complexes is accompanied by an increase of the distance between the O atom of water molecule and the peptide C atom. Additionally, shortening of the peptide C-N bond and decreasing of the distances between the H atom of water molecule and the peptide N atom are observed. The second type of the complexes is characterized by shortening of the OH breaking bond of water molecule and of the O. . .C distances and increasing of the H. . .N distances between water and the peptide in accordance with lowering of the activation energy. The solvent molecule pulls off a proton of reactive water molecule. The shift of electron density to the oxygen atom of water molecule induces shortening of the OH breaking bond and as consequences moving the hydrogen atom apart from the nitrogen atom of dipeptide. The connection between calculated effect of solvent molecules on the activation energy of hydrolytic reaction and the experimental physicochemical parameters of the solvents – donor and acceptor numbers of Gutmann [35] – was analyzed. These parameters are a characteristic of the individual solvent molecule capacity to donate or to accept lone pair electrons. Decreasing of the activation energy in the range of AN – water – alcohols in the complexes of the second type correlates with the donor number of the solvents; DMSO is out of range. The electronegativity of functional groups is a basic characteristic of electron distribution on their atoms and consequently reflects their ability to interact with other molecules. In carboxyl
Fig. 8. Transition states for solvent-assisted mechanism with propionic, pyruvic and lactic acid molecules.
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
acids and alcohols, a greater electronegativity of atom attached to the H–O bond apparently results in a weaker H–O bond, which is thus more readily ionized. The presence of strongly electronegative atoms in acids increases the polarization of the H–O bond facilitating the ionization of the acid and increasing the acid strength. At the same time the presence of electronegative C„N group in acetonitrile molecule gives negative inductive effect to the methyl group and facilitates detaching of methyl proton. We use electronegativity of hydroxyl oxygen atom in carboxyl acids and alcohols to characterize ability of OH group to interact with the reactive water molecule. Electronegativity of methyl carbon is used to characterize the ability of CH3 group of acetonitrile to interact with the reactive water molecule. The electronegativity of the atoms was calculated with account of the inductive effect of the rest part of molecule using the formula [36]:
Eg ¼ ½V c Ec þ
X
Ni Ei =N
where Vc and Ec are the valence of the central atom (the C or hydroxyl O atom) and its electronegativity value, respectively. Ni and Ei are the number of the bond of atoms connected to the central atom and the atomic electronegativity of i atom, respectively, N is the sum of the valence of the central atom and the number of atoms connected to the central atom. Fig. 9 shows dependence of the solvent effect on the activation energy of hydrolytic reaction on the electronegativity of hydroxyl O atom of alcohols and carbon acids and methyl C atom for acetonitrile. The effect of organic solvent on the activation energy was estimated as the difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule; this dif-
289
ference was denoted as relative activation energy. Fig. 9(A) gives the evidence that there exists a correlation between the contribution of solvent in the activation energy and electronegativity of central atom for the first type of complexes of the non-assisted mechanism with correlation coefficient of 0.875. It is found a linear relationship between the influence of the organic molecules on the activation energy of hydrolytic reaction and the electronegativity for complexes of the solvent-assisted mechanism (Fig. 9(B)) with correlation coefficient of 0.995. Electronegativity of central atom of the proton containing functional group is a measure of ability of this group to detach a proton that is reflected by pKa. We plot the effect of the organic molecules on the activation energy versus pKa in Fig. 10. Fig. 10(A) shows a linear relationship of the value of the organic solvent influence on the activation energy in non-assisted mechanism and pKa of the solvent with correlation coefficient of 0.977. Fig. 10(B) demonstrates the correlation of the relative activation energy in solvent assisted mechanism and pKa with correlation coefficient of 0.854.
4. Conclusion We studied the effect of organic compound on the nonenzymatic hydrolytic reactions of the peptide bond cleavage. Dipeptide of alanine was used as a model system. Quantum chemical calculation of the structure of transition states and activation energy was performed at DFT theoretical level using B3LYP density functional and 6-31G(d) basis set. The efficiency of organic solvents in decreasing or increasing the activation energy barrier of reaction was evaluated relative to water effect. The transition states for system alanine dipeptide – reactive water molecule – organic solvent were localized. Several organic solvents possessing different
Fig. 9. Relative activation energy of the peptide hydrolysis vs electronegativity of functional groups for the complexes of the first type of non-assisted mechanism (A) and for solvent assisted mechanism (B). Relative activation energy is calculated as a difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule. Electronegativity of the hydroxyl O atom for the alcohols, acids, and water and electronegativity of the methyl C atom for acetonitrile were used.
Fig. 10. Relative activation energy of the peptide hydrolysis vs pKa of functional groups for the complexes of first type of non-assisted mechanism (A) and for solvent assisted mechanism (B). Relative activation energy is calculated as a difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule.
290
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
physicochemical properties were chosen: methanol, ethanol, 1propanol, acetonitrile (AN), dimethylsulfoxide (DMSO), propionic, lactic, and pyruvic acids. We focused our attention on the concerted catalyzed and non-catalyzed mechanisms of the hydrolytic reaction. In the former mechanism, the solvent molecule is involved in reaction as catalyst and participates in the proton transfer; in the latter, the solvent molecule interacts with the reactive water molecule and has indirect influence on the peptide hydrolysis. It was found that donor–acceptor interactions between dipeptide, reactive water molecule and molecules of solvents revealed strong influence on the activation energy barrier of the peptide bond hydrolysis. Alcohols and DMSO decrease the activation energy in the same order of magnitude as water. Acetonitrile can act as an inhibitor of reaction. Strong influence of organic acids on the activation energy barrier correlates with their electronegativity.
[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
Acknowledgment The support of Russian Academy of Sciences for Basic Research under the program ‘‘Molecular and Cellular Biology’’ and Russian Foundation for Basic Research Grants N 12-04-31360 is acknowledged. Calculations were carried out at Joint Supercomputer Center of Russian Academy of Sciences. Appendix A. Supplementary data
[25] [26] [27] [28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphys. 2013.01.037.
[29] [30]
References
[31] [32] [33]
[1] A.J. Cordoba, B.J. Shyong, D. Breen, R.J. Harris, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 818 (2005) 115. [2] M.A. Smith, M. Easton, P. Everett, G. Lewis, M. Payne, V. Riveros-Moreno, G. Allen, Int. J. Pept. Protein Res. 48 (1996) 48. [3] S.L. Cohen, C. Price, J. Vlasak, J. Am. Chem. Soc. 129 (2007) 6976.
[34] [35] [36]
A.M. Klibanov, Nature 409 (2001) 241. P. Ball, Chem. Rev. 108 (2008) 74. D. Kahne, W.C. Still, J. Am. Chem. Soc. 110 (1998) 7529. A. Radzicka, R. Wolfenden, Science 267 (1995) 90. A. Radzicka, R. Wolfenden, J. Am. Chem. Soc. 118 (1996) 6105. R.M. Smith, D.E. Hansen, J. Am. Chem. Soc. 120 (1998) 8910. J.P. Krug, P.L.A. Popelier, R.F.W. Bader, J. Phys. Chem. 96 (1992) 7604. S. Antonczak, M.F. Ruiz-López, J.L. Rivail, J. Am. Chem. Soc. 116 (1994) 3912. S. Antonczak, M.F. Ruiz-López, J.L. Rivail, J. Mol. Model. 3 (1997) 434. L. Gorb, A. Asensio, I. Tunon, M.F. Ruiz-Lopez, Chem. Eur. J. 11 (2005) 6743. B. Wang, Z. Cao, J. Phys. Chem. A 114 (2010) 12918. S. Tolosa, J.A. Sansón, A. Hidalgo, Chem. Phys. 353 (2008) 73. D. Zahn, Eur. J. Org. Chem. 2004 (2004) 4020. B. Pan, M.S. Ricci, B.L. Trout, J. Phys. Chem. B 114 (2010) 4389. B. Pan, M.S. Ricci, B.L. Trout, J. Phys. Chem. B 115 (2011) 5958. N. Tsuchida, H. Satou, S. Yamabe, J. Phys. Chem. A 111 (2007) 6296. B. Wang, Z. Cao, Chem. Eur. J. 17 (2011) 11919. C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785. A.D. Becke, J. Chem. Phys. 98 (1993) 5648. J.S. Binkley, J.A. Pople, W.J. Hehre, J. Am. Chem. Soc. 102 (1980) 939. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, N.J. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.02 Gaussian Inc., Wallingford, C.T., 2009. J. Tomasi, M. Persico, Review 94 (1994) 2027. J. Tomasi, B. Mennucci, E. Cancis, J. Mol. Struct. 464 (1999) 211. V. Barone, M. Cossi, J. Phys. Chem. A 102 (1998) 1995. J.B. Foresman, T.A. Keith, K.B. Wiberg, J. Snoonian, M.J. Frisch, J. Phys. Chem. 100 (1996) 16098. A.V. Marenich, C.J. Cramer, D.G. Truhlar, J. Phys. Chem. B 113 (2009) 6378. D. Cheshmedzhieva, S. Ilieva, B. Hadjieva, B. Galabov, J. Phys. Org. Chem. 22 (2009) 619. M. Strajbl, J. Florian, A. Warshel, J. Am. Chem. Soc. 122 (2000) 5354. G. Estiu, K.M. Merz Jr., J. Phys. Chem. B 111 (2007) 6507. R.P. Bora, A. Barman, X. Zhu, M. Ozbil, R. Prabhakar, J. Phys. Chem. B 114 (2010) 10860. O. Makshakova, D. Chachkov, E. Ermakova, Int. J. Quantum. Chem. 111 (2011) 2525. V. Gutmann, Electrochim. Acta 21 (1976) 661. H. Wu, Molecules 4 (1999) 16.
Contents lists available at SciVerse ScienceDirect
Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Effect of organic molecules on hydrolysis of peptide bond: A DFT study Olga Makshakova, Elena Ermakova ⇑ Kazan Institute of Biochemistry and Biophysics RAS, 420111 Kazan, P.O. Box 30, Russia
a r t i c l e
i n f o
Article history: Received 2 November 2012 In final form 30 January 2013 Available online 9 February 2013 Keywords: Density functional theory Transition state Dialanine Organic solvent
a b s t r a c t The activation and inhibition effects of small organic molecules on peptide hydrolysis have been studied using a model compound dialanine and DFT approach. Solvent-assisted and non-assisted concerted mechanisms were analyzed. Several transition states for the systems: alanine dipeptide–water molecule in complexes with alcohol molecules, acetonitrile, dimethylsulfoxide, propionic, lactic and pyruvic acids and water molecules were localized. The formation of hydrogen bonds between dipeptide, reactive water molecule and molecules of solvents influences the activation energy barrier of the peptide bond hydrolytic reaction. Strong effect of organic acids on the activation energy barrier correlates with their electronegativity. Acetonitrile can act as an inhibitor of reaction. Mechanisms of regulation of the activation energy barrier are discussed in the terms of donor-acceptor interactions. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction The peptide bond cleavage is a major process of protein decomposition and metabolism of living matter. The covalent stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Both enzymatic and nonenzymatic hydrolysis of peptide bond is of biological and technological significance [1–4]. The reaction mechanism of nonenzymatic peptide hydrolysis has been studied from many points of view [5–19] and is still an object of theoretical investigation by modern calculation methods [10–18]. From the theoretical point of view, the peptide bond has been modeled using different compounds like formamide [10– 15], N-methylacetamide [16–18], N,N-dimethylformamide [19] and the mechanisms of bond cleavage have been treated at different levels of theory, including cluster analysis [10–14] and ab initio CPMD simulations [16–18]. At present four different mechanisms of hydrolysis are discussed: the concerted mechanism with and without the additional water molecule and the stepwise mechanism with and without the additional water molecule. In the concerted mechanism, the addition of the water molecule and cleavage of the CN amide bond occur in a single step [13]. In the stepwise mechanism, water is first added to the CO bond to form a gem-diol intermediate and subsequently the proton transfer from one of the OH groups to the nitrogen atom induces the dissociation of the system and formation of the products. Participation of the second water molecule in each
⇑ Corresponding author. Tel.: +7 843 2319037; fax: +7 843 2927347. E-mail address: [email protected] (E. Ermakova). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.01.037
mechanism (the water-assisted mechanism) decreases the activation energy. According to the findings of Refs. [11–13], nucleophilic attack on carbon atom is the rate limiting step of all reaction mechanism and the energy barriers for the concerted mechanism and the stepwise one are nearly close (1 kcal/mol). CPMD calculations found no intermediate in the reaction dynamics and showed that the concerted attack of carbon and nitrogen atoms by water molecules directly results to products [17]. We believe that the effect of organic solvent molecules at the first stage of the stepwise pathway is qualitatively similar to their effects on the concerted mechanism studied here. The detailed analysis of the stepwise mechanism is out of scope of the present work. Here we only deal with the concerted mechanism. It is well known that the physicochemical properties of environment have crucial impact on the rates of both chemical and biochemical reactions. Different theoretical approaches have been developed over the years to account for water as an essential living environment. Water environment was taken into account both explicitly [13,16–18] or implicitly by continuum models [11,20] or by means of the combined model of solvent. In the combined model the central compound was solvated explicitly by some solvent molecules and the resulting cluster was treated by a dielectric continuum model [14]. Live cells are crowded with high- and low-molecular organic compounds which change the thermodynamic activity of water and can specifically interact with reagents, transition states, intermediates and products of reactions [5]. Such organic molecules as alcohols and acids can regulate the rate of the hydrolytic reaction indirectly as well as interact with peptide inducing a peptide bond breaking. The direct interaction is intensively studied experimentally and theoretically [19].
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Main aim of our investigation was to define an indirect influence of the organic molecules on the peptide bond hydrolysis thorough their interaction with reactive water molecule. In this study we have focused attention on the influence of water molecule and organic solvent molecules on the concerted mechanism of hydrolysis, on the structure and energy of transition state and on the activation energy for nonenzymatic hydrolytic reaction using a model compound, dialanine, and DFT computational method. Several organic solvents with significant technological importance were considered: methanol, ethanol, 1-propanol, acetonitrile (AN), dimethylsulfoxide (DMSO), carboxylic acids (propionic, lactic and pyruvic). To our knowledge, there have not been previously reported systematic investigations of the structural and energetic aspects of the peptide bond hydrolysis in the presence of organic solvent molecules in literature. Understanding of the influence of organic compounds on nonenzymatic hydrolysis reactions can provide a new insight into mechanism of non-aqueous enzymatic reactions where water–organic solvent is used as reaction media [4]. The information is also can be useful for the development of novel drugs based on transition state or intermediate structures. 2. Theoretical approach The initial structure of alanine dipeptide (hereafter dipeptide) was slightly modified by substituting of hydroxyl fragment of carboxyl group of C-terminus and of a hydrogen atom of N-terminus with methyl groups in order to weaken their influence on the studied reaction. The DFT method using hybrid functional B3LYP [21,22] was applied for the location of minima and transition states (TS) structures. The Pople’s split-valence basis set 6-31G [23] was used including the polarization and diffusion functions 6-31G(d), 6-31+G(d, p), 6-31+G(2df, p), 6-31++G(2df, p), and 6311++G(2df, 2p). Structural parameters and energies for reactants and transition states were defined using the program package Gaussian09 [24]. The location of TS structures was performed starting from the dipeptide initial structure and adding a water molecule to the peptide bond. The geometries of all transition states and reactants were fully optimized at corresponding level of theory. The transition state structures were verified by the analysis of the normal vibration frequencies. The correlation between stable structures and transition states was checked by analysis of the corresponding imaginary frequency mode and by the intrinsic reaction coordinate (IRC) calculations. The stationary structures were recognized as true minima after verifying the lack of imaginary frequencies. The activation energy was calculated using the following formula:
E ¼ ETS Epept EW
283
lactic and pyruvic acids were used to study the influence of the explicit solvent on the activation energy barrier. We repeated the search for transition states including one low-weight organic molecule or one extra water molecule. Calculations were performed in gas phase conditions using B3LYP/6-31G(d) basis set. The activation energy was calculated from the formula:
E ¼ ETS Epept EW ES
ð2Þ
where Es is energy of explicit solvent and other designations are the same as in (1). 3. Results and discussion 3.1. Non-assisted mechanism First we have studied the well known concerted non-assisted mechanism of peptide bond hydrolysis to compare our calculations with the results obtained earlier [11,13]. According to this mechanism, single water molecule forms with dipeptide the four-member ring, which was previously described by Antonczak et al. [11] for formamide hydrolysis. Fig. 1 depicts the geometry of the transition state (TS). Table 1S of Supplementary contains some geometry parameters of the transition state. The predicted TS geometry of dialanine is very similar to the geometry of transition state of formamide hydrolysis obtained at the MP2 level with 6311++G(d, p) basis set [15]. The comparison of the activation energies calculated using different basis sets (Table 1) evidences a strong decrease of the energy values of reagents with the increase of the basis set from 6-31G(d) to 6-311++G(2df, 2p). At the same time, the energy values of the transition state are slowly decreased with the basis set augmentation. As a result, one can see the increase of the activation energy barrier of the hydrolytic reaction along with the basis set augmentation. Gorb et al. [13] applied different methods and basis sets for calculation of transition state in the reaction of formamide hydrolysis in water. The comparison of both the activation energies and free energy barriers obtained with B3LYP/6-31G(d) and MP2/6311+G(d, p) revealed a good qualitative agreement between both methods but the B3LYP provided smaller values than MP2. The comparison of the potential energies calculated at different levels and different basis sets (B3LYP/6-31+G(d, p), B3LYP/cc-pVTZ, MP2/cc-pVTZ) was also carried out in Ref. [18]. DE was found in the range from 40.4 to 43.3 kcal/mol depending on the basis set and method.
ð1Þ
where, ETS, Epept and EW are the energy of transition state, dipeptide and water molecule. All energy values were corrected taking into account the zero-point vibrational energies. For TS, zero-point vibrational energy was calculated missing the imaginary frequency. The detailed analysis of implicit influence of bulk solvent was performed for non-assisted mechanism. Four different continuum models were used (polarizable continuum mode (PCM) [25,26], polarizable conductor calculation model (CPCM) [27], isodensity polarizable continuum mode (IPCM) [28], and solvation model of density (SMD) [29] to take into account the effects of bulk water. Only PCM model was used for organic solvents. Dielectric permeability were 78.36, 24.85, 35.69, and 46.83 for water, ethanol, AN, and DMSO, respectively. Molecules of organic solvents: methanol, ethanol, 1-propanol, acetonitrile, DMSO, and molecules of carboxylic acids: propionic,
Fig. 1. Transition state for the gas phase reaction of the peptide hydrolysis in nonassisted mechanism.
284
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Table 1 Energies of the transition and ground states (Hartrees) and the activation energies (kcal/mol) of the peptide bond hydrolysis calculated with B3LYP functional at different basis sets.
ETS Epept Ew DE TDS DG
6-31G(d)
6-31+G(d, p)
6-31+G(2df ,p)
6-31++G(2df, p)
6-311++G(2df, 2p)
650.7902794 574.444677 76.4089533 40 12.5 51
650.8578779 574.4950466 76.4340477 44.8 12.7 56
650.8858427 574.5187152 76.4406146 46.2 12.6 57.4
650.886186 574.5189777 76.4406664 46.2 12.6 57.4
651.0423138 574.6549343 76.4626211 47.2 12.4 58.2
DE = ETS Epept Ew. TDS = T(STS Spept SW). DG = GTS Gpept GW. Subscripts designate: w –water molecule, pept – dipeptide.
It is well known that entropic effects play a crucial role in the reactivity of weakly bonded complexes. Large negative TDS values were obtained for the water assisted and non-assisted formamide hydrolysis with amount 10–12 kcal/mol per water molecule [12]. However, it was demonstrated that entropy terms evaluated from the theoretical computations referring to gas phase processes were significantly overestimated [30,31]. Consequently the calculated free energies were overestimated as well. Different scientific groups reported free energy values ranging from 35 to 60 kcal/mol [11,13,32]. Estiu et al. [32] have showed that the primary origin of the difference between the values reported by different groups was based on the definition of the initial state. The calculation of the free energy barrier relative to the separated reagents (see Theoretical approach) with 6-311++G(2df, 2p) basis set gives 58 kcal/mol (Table 1), that is very close to results of Estiu et al. [32] obtained with MP2/6-311++G⁄⁄ level of theory. Taking into account the solvent effect as a continuum producing the polarization of solute we can evaluate the media effect on molecular complexes. We compared several continuum solvent models to estimate the role of environmental water in stabilization of the transition state of the dipeptide hydrolysis. Four different continuum models demonstrate the similar results (Table 2S). The comparison of main geometry parameters of TS reveals a small increase of the length of OH breaking bond and a decrease of the CN peptide bond for three continuum models (PCM, CPCM, and SMD). The distance between the O atom of water molecule and the C atom of peptide slightly elongates and the distance between the H atom of water and the N atom of peptide shortens (Table 2S). The implicit account of environmental water reduces the energy of both the reagents and the TS. Taking account of bulk water by PCM decreases the transition state energy by about 10 kcal/mol. At the same time the energies of the dipeptide and water molecule are reduced by 7.5 and 4.5 kcal/mol, respectively, compared with the results in the gas phase. Thereby the activation energy growth by about 2 kcal/mol is observed. SMD gives the lowest values of the energy of both the reagents and the TS. SMD stabilizes TS, peptide and water molecule by 19, 12, and 8.2 kcal/mol, respectively, compared with the results in the gas phase. Implicitly treated solvents demonstrated rather small effect in Ref. [13], the largest change in DG was less than 2 kcal/mol. This is in agreement with the previous suggestion made by Antonczak et al. [11]. Similar slight effect of continuum solvent was found in the recent study of enzymatic peptide hydrolysis [33]. Unfortunately PCM demonstrates no effect of solution dielectric constant on the activation energy. The activation energy for all used organic solvents, 41.7 kcal/mol, is very similar to the result found for bulk water (Table 3S). Comparing the results obtained with different basis sets and different dielectric continuum solvent models we conclude that implicitly treated solvents demonstrate rather small effect on the activation energy and B3LYP method with small basis set
6-31G(d) slightly underestimates the activation energy barrier, but it gives a better agreement with the experimental data [7,8]. Additionally, for large biomolecule or for ab initio MD studies 631G(d) basis set is considered to be the best compromise of speed and accuracy, and is the most commonly used [33]. 3.2. Influence of organic molecule on the transition state energy in non-assisted mechanism Here we focus attention on one point of hydrolysis in a complicated system of peptide, water and organic compounds where the organic molecules influence the water molecule – peptide interactions by means of hydrogen bond formation with the reactive water molecule. The hydrogen bonding complexes of two types were taken into account. In complexes of the first type, an organic molecule acts as a lone pair electron acceptor and interacts with the oxygen atom of water molecule. In complexes of the second type, an organic molecule acts as a lone pair electron donor and interacts with a hydrogen atom of water molecule. Keeping in mind that the entropy contributions to free energy barriers are strongly overestimated compared to the experiment [30,31] we will further discuss the influence of solvent molecule on the activation energy. Calculated values of free energy are also presented in the tables. The first structure (TS_w_C1) possesses an additional water molecule bound to the O atom of reactive water molecule (Fig. 2, Table 4S). The energetic characteristics of the transition state are listed in Table 2. The activation energy barrier for this complex is 32.8 kcal/mol. Since we calculate the energy barrier relative to the separated reactive molecules, the formation of hydrogen bond between two water molecules makes difficult direct comparison of the energy barriers of TS_w_C1 and TS. In the second structure (TS_w_C2) an additional water molecule forms two hydrogen bonds – the O atom is bound to the H atom of reactive water molecule and the H atom of the additional water molecule is bound to the carboxyl O atom of peptide group (Fig. 2). In this ring-like structure the network of several covalent and hydrogen bonds forms a closed structure. Two hydrogen bonds (TS_w_C2 complex) lower the activation energy down to 29.6 kcal/ mol. The influence of methanol, ethanol and propanol molecules on the TS of hydrolysis reaction was investigated (Table 4S). The interaction of alcohol molecule with peptide and water molecule slightly changes the geometry parameters of TS and the activation energy. Two types of different complexes were found for all alcohols. For the first type of complexes (TS_MeOH_C1, TS_EtOH_C1, TS_PrOH_C1), the hydrogen bond formed between the oxygen atom of water molecule and the hydrogen atom of the hydroxyl group of alcohol molecules decreases the energy of the transition state. Interactions of the type of C–H. . .O and C–H...N give an
285
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Fig. 2. Transition states for non-assisted mechanism with additional water molecule.
Table 2 Activation energies (kcal/mol) of the peptide bond hydrolysis in the complexes with water molecule and alcohol molecules in non-assisted concerted mechanism.
DE TDS DG
w_C1
w_C2
MeOH_C1
MeOH_C2
EtOH_C1
EtOH_C2
PrOH_C1
PrOH_C2
32.8 21.6 52.4
29.6 23.1 50.3
30.7 22.5 51.7
27.5 23.4 49.1
30.6 22.4 51.7
27.7 23.6 49.6
30.5 22.6 51.8
27.6 23.3 49.3
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
additional stabilization of complex TS. The importance of weak hydrogen bonds of the type of C–H. . .O and C–H...N is detailed discussed elsewhere [34]. The formation of these complexes leads to very similar activation energies – 30.7, 30.6, 30.5 kcal/mol for methanol, ethanol, and propanol, respectively (Fig. 3, Table 2). The second type of the transition states in complexes with alcohol molecules (TS_MeOH_C2, TS_EtOH_C2, TS_PrOH_C2) is stabilized by two hydrogen bonds forming ring-like structure, respectively. The activation energy of these complexes drops down
to 27.5, 27.7, and 27.6 kcal/mol, for methanol, ethanol, and propanol, respectively. Structures of the complexes of dipeptide with alcohol molecules are similar to the structures of appropriate complexes with water molecule TS_w_C1 and TS_w_C2. The activation energies of dipeptide – water – alcohol systems are similar to each other and are slightly lower than values in dipeptide – water – water systems. Aprotic organic solvent molecules – acetonitrile (AN) and dimethylsulfoxide (DMSO) – have a different influence on the TS
Fig. 3. Transition states for non-assisted mechanism with additional methanol, ethanol and 1-propanol molecules.
286
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
of hydrolysis. Two different transition states for the system of AN – peptide – water were found where AN molecule interacted with water molecule and with peptide by both methyl group and the N atom. In the first complex (TS_AN_C1), AN molecule interacts with water by the methyl group (Fig. 4, Table 5S) and forms two weak bonds between the H atoms of AN and the O atom of water molecule and the N atom of N-terminus of dipeptide. The activation energy barrier for the first type of hydrolysis reaction is higher and is equal to 35.6 kcal/mol (Fig. 4, Table 3). In the second complex (TS_AN_C2), acetonitrile molecule interacts with water molecule by the nitrogen atom and forms the N. . .HOH hydrogen bond between the N atom of AN and the hydrogen atom of water. The second weak hydrogen bond of type CH. . .O is formed between the hydrogen atom of the methyl group of AN and the carbonyl O atom of dipeptide. The activation energy barrier is lower than one of TS_AN_C1 and equals 33.6 kcal/mol (Table 3). One can see that AN weakly interacts with reactants and demonstrates a higher activation energy barrier of the reaction. Dimethylsulfoxide has a strong influence on the peptide hydrolysis as well. Two transition states for the system of DMSO – peptide – water were found where molecule of DMSO interacted with water molecule by the O atom and by the S atom (Fig. 5, Table 5S). In the first TS_DMSO_C1 complex the S atom of DMSO is bound to the H atom of the reactive water molecule, the O atom is bound to the H atom of methyl group of the peptide, and weak hydrogen bond of type CH. . .O is formed between the H atom of the methyl group of DMSO and the O atom of the peptide. This complex is less energetically favorable than TS_DMSO_C2. The formation of this complex results in the activation energy of 31.5 kcal/mol (Table 3). In more favorable TS_DMSO_C2 transition state, one hydrogen bond between the O atom of DMSO and the H atom of water is formed. Two weak bonds between the H atoms of the methyl groups of DMSO and the O atom of peptide group and the N atom of N-terminus of the peptide give additional stabilization for this TS. The formation of this complex leads to the lowering of the activation energy down to 26.3 kcal/mol. 3.3. Effect of carboxylic acid molecules on the transition state energy Three weak acids with different electronegativity were chosen to study the influence of carboxylic acid on the peptide bond hydrolysis. Propionic acid (PrA) is a carboxylic acid with chemical formula CH3CH2COOH. Lactic acid (LA) with the chemical formula CH3CHOHCOOH has a hydroxyl group adjacent to the carboxyl group. Pyruvic acid (PA) (CH3COCOOH) contains oxygen atom at the second carbon position. Electronegative substituents make acids stronger. Electronegativity of these acids increases from propionic acid to lactic and to pyruvic acid. Carboxylic acids are typically weak acids, implying that they only partially dissociate into
Table 3 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with AN and DMSO molecules in non-assisted concerted mechanism.
DE TDS DG
AN_C1
AN_C2
DMSO_C1
DMSO_C2
35.6 20.5 55.2
33.6 21.7 54.2
31.5 23.3 53.7
26.3 23.9 48.8
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
H + cations and RCOO–anions in neutral aqueous solution. We have taken into account only nonionized states of carboxylic acid molecules. Structures of complexes with lactic acid are given in Fig. 6. Geometric parameters and energy of the complexes are given in Table 6S and Table 4. For carboxylic acid molecules in nonionized state, we treated two types of complexes similar to those with water, alcohol, and aprotic organic solvent molecules. Thus, in the complexes of the first type (TS_PrA_C1, TS_PA_C1, TS_LA_C1), the oxygen atom of water molecule forms a strong hydrogen bond with hydroxyl group of carboxyl fragment of acid molecule. The second slight hydrogen bond is formed between the hydrogen atom of water and the carbonyl oxygen atom of acid molecule. Activation energies are equal to 26.9, 24.8 and 24.8 kcal/ mol for propionic, lactic and pyruvic acids, respectively. In the complexes of the second type (TS_PrA_C2, TS_PA_C2, TS_LA_C2), the hydrogen atom of water molecule creates hydrogen bound to the carbonyl oxygen of carboxylic acid molecule. Additional hydrogen bond is formed between the hydrogen atom of carboxyl group of the acid molecule and the carbonyl oxygen of the dipeptide. This type of the complexes is more favorable than the previous one; the energies are 23.2, 23 and 23.1 kcal/mol for PrA, LA and PA, respectively. 3.4. Solvent molecule-assisted mechanism If two water molecules participate in the bond cleavage, such reaction is referred to as water-assisted pathway. In this reaction the nucleophilic attack of one water molecule (W1) on the carbonyl carbon of peptide is accompanied by the concerted proton transfer of the second water molecule (W2) to the nitrogen atom of peptide (Fig. 7). The geometry of TS2_w_C1 is very similar to geometry of the transition state of water-assisted hydrolysis of formamide molecule obtained by Tolosa et al. with 6-311++G⁄⁄ basis set [15]. In the transition state TS2_w_C1 of water-assisted process, a hydrogen atom of the first water molecule (that performs nucleophylic attack) is situated between two oxygen atoms belonging to W1 water molecule and W2 molecule, the distances
Fig. 4. Transition states for non-assisted mechanism with additional acetonitrile molecule.
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
287
Fig. 5. Transition states for non-assisted mechanism with DMSO molecule.
Fig. 6. Transition states for non-assisted mechanism with lactic acid molecule in neutral form.
Table 4 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with carboxyl acid molecules in non-assisted concerted mechanism.
DE TDS DG
PrA_C1
PrA_C2
LA_C1
LA_C2
PA_C1
PA_C2
26.9 21.8 47.6
23.2 24.2 45.9
24.8 21.9 45.6
23 24.3 44.8
24.8 21.8 45.5
23.1 23.9 45.5
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule
H. . .O are 1.203 and 1.219 Å, respectively (Table 7S). The OH bond of W2 water molecule is elongated up to 1.347 Å. Transition state of water-assisted hydrolysis TS2_w_C1 is accompanied by lowering the activation energy down to 25.9 kcal/mol (Table 5). This value is in good agreement with previous results for water-assisted hydrolysis of formamide. The activation energy of formamide hydrolysis was found to reduce by 9 kcal/mol for the assisted concerted mechanism [13]. Further, one can note that the activation energy of TS2_w_C1 complex is lower than non-assisted reaction with the additional water molecule TS_w_C1 and TS_w_C2. In solvent-assisted mechanism, the nucleophilic attack is similar to water-assisted concerted mechanism; solvent molecule plays a role of the second water molecule. Transfer of the proton to the nitrogen atom of dipeptide occurs with the aid of catalytic solvent molecule. Solvent molecule accepts the hydrogen atom from water
molecule and donates its own proton to the nitrogen atom of peptide. Transition states corresponding to alcohol-assisted hydrolytic process are more energetically favorable than ones involved in water-assisted pathway by 4–5 kcal/mol. Hydrogen atom abstracted from water molecule is shifted to the proton acceptor atom of catalytic alcohol molecule; at the same time the OH bond of alcohol molecule elongates up to 1.31–1.32 Å. Three investigated alcohols give similar TSs and the activation energy barriers (Table 7S, Fig. 7). It is known that acetonitrile molecule is able to donate proton of its methyl group because of the strong electronegativity of C„N group. The transition state structure TS2_AN is given in Fig. 7. Methyl group of AN accepts a proton of water molecule and donates one to the N atom of peptide bond. The methyl C–H bond strongly elongates from 1.094 up to 2.264 Å. In AN-assisted mechanism the activation energy barrier significantly increases up to 51.8 kcal/mol. In other words, AN acts as an inhibitor of reaction. The studied carboxylic acids acting as catalysts lower the activation energy down to 14–16 kcal/mol. The hydrogen atom abstracted from water molecule is shifted to the oxygen atom of acid molecule and the OH bond of acid molecule elongates up to 1.57–1.64 Å. The activation energies increase in the range of lactic, pyruvic and propionic acids and are equal to 14.0, 14.2 and 16 kcal/ mol, respectively. (Table 7S, Fig. 8). The action of carboxylic acid in the concerted mechanism is similar to the hydrolysis mechanism of aspartyl proteases which was studied in Ref. [33] and refs therein. The activation energy obtained relative to pre-reaction complex was 22 kcal/mol and
288
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
Fig. 7. Transition states for solvent-assisted mechanism with water molecule, methanol, and acetonitrile.
Table 5 Activation energies (kcal/mol) of the peptide bond hydrolysis in complexes with water and organic molecules in solvent-assisted concerted mechanism.
DE TDS DG
w
MeOH
EtOH
PrOH
AN
PrA
LA
PA
25.9 23.85 46.8
21.3 23.8 43.2
21.3 24.3 43.8
21 23.9 43.3
51.8 24.8 74.8
16 24.8 39.2
14 25.2 37.6
14.2 24.6 37.3
DE = ETS Epept Ew ES. TDS = T(STS Spept Sw SS). DG = GTS Gpept Gw GS. E and G include ZPVE correction. w –water molecule, pept – dipeptide, S – solvent molecule.
should be compared with the free energy barriers obtained here which were in the range from 35 to 39 kcal/mol. It is not surprising that organic molecules act less effectively than proteins. 3.5. Comparison of the influence of organic compounds on the activation energies and on geometry parameters of the transition state In non-assisted mechanism of the peptide hydrolysis, organic compounds demonstrate a different effect on the activation energy of the reaction. Comparing with the additional water molecule contribution, organic molecules can decrease or increase the activation energy of the reaction. Molecules of three studied alcohols decrease the activation energy by 2 kcal/mol, carboxyl acids decrease it by 6–8 kcal/mol, molecule of AN increases the barrier by 4 kcal/mol. Molecule of DMSO decreases the activation energy by 1 kcal/mol. The changes of the activation energy barrier are accompanied by the modifications of geometry parameters of transition state. The discussed complexes can be divided into two groups. The first group includes the complexes where solvent molecule interacts by its H atom with the O atom of reactive water molecule or in other words it acts as the acceptor of the lone pair electrons. The complexes labeled above as C1 (except TS_DMSO_C1) belong to the
first group. The second group contains the complexes where the solvent molecule interplays by its O atom with the H atom of reactive water molecule and donates the lone pair electrons. The complexes of these two groups influence the geometry parameters of the transition state contrariwise. The length of the OH breaking bond of water molecule elongates along with the lowering of the activation energy in the first group of complexes. This finding is in agreement with the theory of the donor-acceptor interactions. The oxygen atom of water molecule donates the lone pair of electrons to solvent molecule. The electron density of the OH bond shifts to the water oxygen atom and induces the elongation of breaking OH bond. The formation of the complexes is accompanied by an increase of the distance between the O atom of water molecule and the peptide C atom. Additionally, shortening of the peptide C-N bond and decreasing of the distances between the H atom of water molecule and the peptide N atom are observed. The second type of the complexes is characterized by shortening of the OH breaking bond of water molecule and of the O. . .C distances and increasing of the H. . .N distances between water and the peptide in accordance with lowering of the activation energy. The solvent molecule pulls off a proton of reactive water molecule. The shift of electron density to the oxygen atom of water molecule induces shortening of the OH breaking bond and as consequences moving the hydrogen atom apart from the nitrogen atom of dipeptide. The connection between calculated effect of solvent molecules on the activation energy of hydrolytic reaction and the experimental physicochemical parameters of the solvents – donor and acceptor numbers of Gutmann [35] – was analyzed. These parameters are a characteristic of the individual solvent molecule capacity to donate or to accept lone pair electrons. Decreasing of the activation energy in the range of AN – water – alcohols in the complexes of the second type correlates with the donor number of the solvents; DMSO is out of range. The electronegativity of functional groups is a basic characteristic of electron distribution on their atoms and consequently reflects their ability to interact with other molecules. In carboxyl
Fig. 8. Transition states for solvent-assisted mechanism with propionic, pyruvic and lactic acid molecules.
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
acids and alcohols, a greater electronegativity of atom attached to the H–O bond apparently results in a weaker H–O bond, which is thus more readily ionized. The presence of strongly electronegative atoms in acids increases the polarization of the H–O bond facilitating the ionization of the acid and increasing the acid strength. At the same time the presence of electronegative C„N group in acetonitrile molecule gives negative inductive effect to the methyl group and facilitates detaching of methyl proton. We use electronegativity of hydroxyl oxygen atom in carboxyl acids and alcohols to characterize ability of OH group to interact with the reactive water molecule. Electronegativity of methyl carbon is used to characterize the ability of CH3 group of acetonitrile to interact with the reactive water molecule. The electronegativity of the atoms was calculated with account of the inductive effect of the rest part of molecule using the formula [36]:
Eg ¼ ½V c Ec þ
X
Ni Ei =N
where Vc and Ec are the valence of the central atom (the C or hydroxyl O atom) and its electronegativity value, respectively. Ni and Ei are the number of the bond of atoms connected to the central atom and the atomic electronegativity of i atom, respectively, N is the sum of the valence of the central atom and the number of atoms connected to the central atom. Fig. 9 shows dependence of the solvent effect on the activation energy of hydrolytic reaction on the electronegativity of hydroxyl O atom of alcohols and carbon acids and methyl C atom for acetonitrile. The effect of organic solvent on the activation energy was estimated as the difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule; this dif-
289
ference was denoted as relative activation energy. Fig. 9(A) gives the evidence that there exists a correlation between the contribution of solvent in the activation energy and electronegativity of central atom for the first type of complexes of the non-assisted mechanism with correlation coefficient of 0.875. It is found a linear relationship between the influence of the organic molecules on the activation energy of hydrolytic reaction and the electronegativity for complexes of the solvent-assisted mechanism (Fig. 9(B)) with correlation coefficient of 0.995. Electronegativity of central atom of the proton containing functional group is a measure of ability of this group to detach a proton that is reflected by pKa. We plot the effect of the organic molecules on the activation energy versus pKa in Fig. 10. Fig. 10(A) shows a linear relationship of the value of the organic solvent influence on the activation energy in non-assisted mechanism and pKa of the solvent with correlation coefficient of 0.977. Fig. 10(B) demonstrates the correlation of the relative activation energy in solvent assisted mechanism and pKa with correlation coefficient of 0.854.
4. Conclusion We studied the effect of organic compound on the nonenzymatic hydrolytic reactions of the peptide bond cleavage. Dipeptide of alanine was used as a model system. Quantum chemical calculation of the structure of transition states and activation energy was performed at DFT theoretical level using B3LYP density functional and 6-31G(d) basis set. The efficiency of organic solvents in decreasing or increasing the activation energy barrier of reaction was evaluated relative to water effect. The transition states for system alanine dipeptide – reactive water molecule – organic solvent were localized. Several organic solvents possessing different
Fig. 9. Relative activation energy of the peptide hydrolysis vs electronegativity of functional groups for the complexes of the first type of non-assisted mechanism (A) and for solvent assisted mechanism (B). Relative activation energy is calculated as a difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule. Electronegativity of the hydroxyl O atom for the alcohols, acids, and water and electronegativity of the methyl C atom for acetonitrile were used.
Fig. 10. Relative activation energy of the peptide hydrolysis vs pKa of functional groups for the complexes of first type of non-assisted mechanism (A) and for solvent assisted mechanism (B). Relative activation energy is calculated as a difference between the activation energy of the complexes with organic solvents and the activation energy of the corresponding complex with additional water molecule.
290
O. Makshakova, E. Ermakova / Chemical Physics 415 (2013) 282–290
physicochemical properties were chosen: methanol, ethanol, 1propanol, acetonitrile (AN), dimethylsulfoxide (DMSO), propionic, lactic, and pyruvic acids. We focused our attention on the concerted catalyzed and non-catalyzed mechanisms of the hydrolytic reaction. In the former mechanism, the solvent molecule is involved in reaction as catalyst and participates in the proton transfer; in the latter, the solvent molecule interacts with the reactive water molecule and has indirect influence on the peptide hydrolysis. It was found that donor–acceptor interactions between dipeptide, reactive water molecule and molecules of solvents revealed strong influence on the activation energy barrier of the peptide bond hydrolysis. Alcohols and DMSO decrease the activation energy in the same order of magnitude as water. Acetonitrile can act as an inhibitor of reaction. Strong influence of organic acids on the activation energy barrier correlates with their electronegativity.
[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]
Acknowledgment The support of Russian Academy of Sciences for Basic Research under the program ‘‘Molecular and Cellular Biology’’ and Russian Foundation for Basic Research Grants N 12-04-31360 is acknowledged. Calculations were carried out at Joint Supercomputer Center of Russian Academy of Sciences. Appendix A. Supplementary data
[25] [26] [27] [28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphys. 2013.01.037.
[29] [30]
References
[31] [32] [33]
[1] A.J. Cordoba, B.J. Shyong, D. Breen, R.J. Harris, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 818 (2005) 115. [2] M.A. Smith, M. Easton, P. Everett, G. Lewis, M. Payne, V. Riveros-Moreno, G. Allen, Int. J. Pept. Protein Res. 48 (1996) 48. [3] S.L. Cohen, C. Price, J. Vlasak, J. Am. Chem. Soc. 129 (2007) 6976.
[34] [35] [36]
A.M. Klibanov, Nature 409 (2001) 241. P. Ball, Chem. Rev. 108 (2008) 74. D. Kahne, W.C. Still, J. Am. Chem. Soc. 110 (1998) 7529. A. Radzicka, R. Wolfenden, Science 267 (1995) 90. A. Radzicka, R. Wolfenden, J. Am. Chem. Soc. 118 (1996) 6105. R.M. Smith, D.E. Hansen, J. Am. Chem. Soc. 120 (1998) 8910. J.P. Krug, P.L.A. Popelier, R.F.W. Bader, J. Phys. Chem. 96 (1992) 7604. S. Antonczak, M.F. Ruiz-López, J.L. Rivail, J. Am. Chem. Soc. 116 (1994) 3912. S. Antonczak, M.F. Ruiz-López, J.L. Rivail, J. Mol. Model. 3 (1997) 434. L. Gorb, A. Asensio, I. Tunon, M.F. Ruiz-Lopez, Chem. Eur. J. 11 (2005) 6743. B. Wang, Z. Cao, J. Phys. Chem. A 114 (2010) 12918. S. Tolosa, J.A. Sansón, A. Hidalgo, Chem. Phys. 353 (2008) 73. D. Zahn, Eur. J. Org. Chem. 2004 (2004) 4020. B. Pan, M.S. Ricci, B.L. Trout, J. Phys. Chem. B 114 (2010) 4389. B. Pan, M.S. Ricci, B.L. Trout, J. Phys. Chem. B 115 (2011) 5958. N. Tsuchida, H. Satou, S. Yamabe, J. Phys. Chem. A 111 (2007) 6296. B. Wang, Z. Cao, Chem. Eur. J. 17 (2011) 11919. C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785. A.D. Becke, J. Chem. Phys. 98 (1993) 5648. J.S. Binkley, J.A. Pople, W.J. Hehre, J. Am. Chem. Soc. 102 (1980) 939. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.J.A. Montgomery, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, N.J. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.02 Gaussian Inc., Wallingford, C.T., 2009. J. Tomasi, M. Persico, Review 94 (1994) 2027. J. Tomasi, B. Mennucci, E. Cancis, J. Mol. Struct. 464 (1999) 211. V. Barone, M. Cossi, J. Phys. Chem. A 102 (1998) 1995. J.B. Foresman, T.A. Keith, K.B. Wiberg, J. Snoonian, M.J. Frisch, J. Phys. Chem. 100 (1996) 16098. A.V. Marenich, C.J. Cramer, D.G. Truhlar, J. Phys. Chem. B 113 (2009) 6378. D. Cheshmedzhieva, S. Ilieva, B. Hadjieva, B. Galabov, J. Phys. Org. Chem. 22 (2009) 619. M. Strajbl, J. Florian, A. Warshel, J. Am. Chem. Soc. 122 (2000) 5354. G. Estiu, K.M. Merz Jr., J. Phys. Chem. B 111 (2007) 6507. R.P. Bora, A. Barman, X. Zhu, M. Ozbil, R. Prabhakar, J. Phys. Chem. B 114 (2010) 10860. O. Makshakova, D. Chachkov, E. Ermakova, Int. J. Quantum. Chem. 111 (2011) 2525. V. Gutmann, Electrochim. Acta 21 (1976) 661. H. Wu, Molecules 4 (1999) 16.