ARTICLE pubs.acs.org/JPCB
Quantum Theoretical Study of Cleavage of the Glycosidic Bond of 20-Deoxyadenosine: Base Excision-Repair Mechanism of DNA by MutY Saumya Tiwari,† Neha Agnihotri, and P.C. Mishra* Department of Physics, Banaras Hindu University, Varanasi 221005, India
bS Supporting Information ABSTRACT: The enzyme adenine DNA glycosylase, also called MutY, is known to catalyze base excision repair by removal of adenine from the abnormal 20 -deoxyadenosine:8-oxo-20 deoxyguanosine pair in DNA. The active site of the enzyme was considered to consist of a glutamic acid residue along with two water molecules. The relevant reaction mechanism involving different barrier energies was studied theoretically. Molecular geometries of the various molecules and complexes involved in the reaction, e.g., the reactant, intermediate, and product complexes as well as transition states, were optimized employing density functional theory at the B3LYP/6-31G(d,p) level in the gas phase. It was followed by single-point energy calculations at the B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-cc-pVDZ, and MP2/AUG-ccpVDZ levels in the gas phase. Single-point energy calculations were also carried out at the B3LYP/AUG-cc-pVDZ and BHandHLYP/AUG-cc-pVDZ levels in aqueous media as well as in the solvents chlorobenzene and dichloroethane. For the solvation calculations, the integral equation formalism of the polarizable continuum model (IEF-PCM) was employed. It is found that glutamic acid along with two water molecules would effectively cleave the glycosidic bond of adenosine by a new two-step reaction mechanism proposed here which is different from the three-step mechanism proposed by other authors earlier regarding the working mechanism of MutY.
1. INTRODUCTION A large number of endogenous and exogenous reactive oxygen species, reactive nitrogen oxide species, alkylating agents, and radiation can damage DNA by producing a wide variety of modifications of its constituents, particularly the bases.1-10 These modifications act as lesions, are hazardous to normal cell functioning, and can cause several lethal effects including mutation, aging, and cancer. The reactive oxygen species and reactive nitrogen oxide species can damage biomolecules including DNA, RNA, proteins, and lipids.11-13 One of the common and lethal lesions thus produced is 8-oxo-20 -deoxyguanosine (OG) from 20 -deoxyguanosine.14 OG mispairs with 20 -deoxyadenosine (dAdo) and can cause mutation.8-11 It is known that antioxidants can scavenge many types of reactive species present in biological media and thus prevent DNA damage.15,16 DNA repair mechanisms belong to two categories, i.e., direct repair mechanism and indirect repair mechanism. In the direct repair mechanism, the damage is simply reversed, while in the indirect repair mechanism, a cutting and patching process that restores normal base pairing is involved.17 An example of the direct DNA repair mechanism is the retrieval of guanine from O6-methylguanine by the enzyme O6-alkylguanine-DNA alkyltransferase (AGT),18-20 while an example of an indirect DNA repair mechanism is base excision repair catalyzed by the enzyme MutY. The main role of MutY, also called adenine DNA glycosylase, is to prevent guanine (G) to thymine (T) transversion that follows OG:dAdo mispairing in DNA.21,22 MutY initiates a repair pathway in which the glycosidic bond of adenosine of the OG: dAdo mismatches is cleaved, and ultimately the normal base r 2011 American Chemical Society
pairing is restored.23-30 The cell has several lines of defense against direct lesion formation that includes different repair proteins which are lesion-specific DNA glycosylases.31,32 The so-called monofunctional glycosylases are hydrolase enzymes that use a water molecule to attack the anomeric carbon of the damaged nucleotide, whereas those of the other type called bifunctional glycosylases use an amine group in place of a water molecule.33-37 MutY is a monofunctional glycosylase, whereas another enzyme of the same family called MutM is a bifunctional glycosylase. There are two steps involved in the base excision repair, the first step being flipping of the damaged base from the DNA duplex into the enzyme active site, while the second step is the catalytic glycosidic bond cleavage.23,30,35 Several crystal structures of DNA glycosylases have been studied, and a great deal of experimental work has been carried out to elucidate the enzymatic mechanisms of glycosidic bond cleavage.24-27 A number of computational studies have also been carried out on this problem.23,35-41 Calvaresi et al. have theoretically investigated glycosylase catalytic activity of human DNA repair protein hOGG1 using density functional theory (DFT).34 They showed that the enzyme causes cleavage of the glycosidic bond and removal of the damaged base. Rios-Font et al.38,39 have studied the mechanism of glycosidic bond hydrolysis in 20 deoxyguanosine involving a single water molecule using DFT. Received: November 16, 2010 Revised: January 26, 2011 Published: March 08, 2011 3200
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Figure 1. Structures of two conformers of each of dAdo (a: target and b: typical) and glutamic acid (c: GluI and d: GluII). The relative Gibbs free energies (kcal/mol) of (a) and (b) obtained at the MP2/AUG-cc-pVDZ level of theory in the gas phase using the geometries optimized at the B3LYP/631G(d,p) level in the gas phase are given with respect to that of (a), while the relative Gibbs free energies of (c) and (d) obtained at the MP2/AUG-ccpVDZ level of theory in the gas phase are given with respect to that of (c).
They showed that N7-protonation of guanine catalyzes hydrolysis of the glycosidic bond. Millen et al.35 have studied the glycosidic bond cleavage in deoxyuridine using DFT. Fromme et al.25 have reported use of disulfide cross-linking to obtain the high-resolution crystal structures of MutY-DNA lesion recognition complexes. Their study reveals the basis for recognizing lesions in the OG:dAdo pair and for catalyzing removal of the adenine base.25 On the basis of the crystal structure of Escherichia coli MutY (eMutY) using multiple kinetic isotope effect measurements, transition state structures (TSs) of MutY-catalyzed DNA hydrolysis were obtained by McCann and Berti.28 These authors also studied the energetics of the reaction mechanism proposed by them employing density functional theory at the B3PW91/631þG(d,p) level. However, no independent and detailed theoretical study of the reaction mechanism yielding transition state, intermediate structures, and barrier energies has yet been reported. Such a theoretical study is highly desirable for validation of the proposed mechanism28 or for incorporation of appropriate changes in the same. For this reason, we have studied the MutYcatalyzed cleavage of the glycosidic bond of 20 -deoxyadenosine (dAdo) using reliable theoretical methods.
2. COMPUTATIONAL DETAILS The active site of MutY was considered to be represented by glutamic acid along with two water molecules, as done previously.28 Molecular geometries of two conformers of dAdo, called target and typical dAdo, and those of two conformers of
glutamic acid (GluI and GluII) (Figure 1) were fully optimized using the B3LYP functional of DFT in the gas phase.42-44 The differences between the typical and target dAdo will be discussed in section 3.1. Geometries of reactant complexes (RC1 and RC2), intermediate complexes (IC1 and IC2), transition states (TS1, TS2, and TS3), and the product complex (PC) involved in the reaction of target dAdo with the conformer GluI of glutamic acid were optimized at the B3LYP/6-31G(d,p) level of theory in the gas phase. Single-point energy calculations for all the optimized geometries of the molecules and complexes were performed at the B3LYP/AUG-cc-pVDZ, BHandHLYP/AUGcc-pVDZ, and MP2/AUG-cc-pVDZ levels of theory in the gas phase.45-48 The optimized structures were solvated in aqueous media by performing single-point energy calculations at the B3LYP/AUG-cc-pVDZ and BHandHLYP/AUG-cc-pVDZ levels of theory using the geometries optimized at the B3LYP/631G(d,p) level in the gas phase and the integral equation formalism of the polarizable continuum model (IEF-PCM).49-51 It has been reported that solvation in protein environments can be treated more accurately by considering the solvent medium as having the dielectric constant (ε) lying between 2 and 10, particularly chlorobenzene that has dielectric constant 5.6, than in water (ε = 78.4).52-55 Due to this reason, solvation of the various species was also studied in this work in chlorobenzene beside water. Further, as convergence failure was encountered in the solvation calculations in chlorobenzene for one case (TS2), solvation calculations were also performed in another solvent with a moderate dielectric constant, i.e., dichloroethane (ε = 10.4). The BHandHLYP functional of DFT was employed here 3201
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Table 1. Relative Gibbs Free Energies (kcal/mol) of the Two Conformers of Each of dAdo (Target and Typical dAdo) and Glutamic Acid (Glu I and Glu II) Obtained at Different Levels of Theory in Different Mediaa,b B3LYP/
BHandHLYP/
MP2/
6-31G(d,p)
AUG-cc-pVDZ
AUG-cc-pVDZ
ε = 5.6d
ε = 78.4e
0.0
0.0
0.0
0.0
0.5
0.4
0.3
1.6
Glu I
0.0
0.0
0.0
0.0
Glu II
2.2
0.7
0.5
1.0
conformer
ε = 1c
target dAdo typical dAdo
ε = 1c
a
The Gibbs free energies of target and typical dAdo are given with respect to that of target dAdo, while those of Glu I and Glu II are given with respect to that of Glu I. b Geometry optimization was performed at the B3LYP/6-31G(d,p) level in the gas phase, while single-point energy calculations were performed at the other two levels of theory. c ε = 1.0 (gas phase). d ε = 5.6 (chlorobenzene). e ε = 78.4 (water).
as it has been shown to be more reliable in some cases than even the popular functional B3LYP.6,56-58 Electrostatic potentialfitted point charges located at the atomic sites were obtained using the CHelpG algorithm59 at the MP2/AUG-cc-pVDZ level of theory in the gas phase. Vibrational frequency analysis was performed for each optimized structure at the B3LYP/6-31G(d,p) level of theory in the gas phase to ensure that each searched structure extremum was genuine, each minimum having all real vibrational frequencies and each transition state having only one imaginary frequency. Zero-point energy (ZPE)-corrected total energies and Gibbs free energies at 298.15 K were obtained in each case at the B3LYP/631G(d,p) level of theory in the gas phase. As an approximation, the ZPE corrections and thermal energy corrections giving Gibbs free energies obtained at the B3LYP/6-31G(d,p) level were also applied to the total energies obtained by single-point energy calculations in the gas phase and different solutions at all the other levels of theory employed here. The genuineness of transition states was ensured by visually examining the vibrational modes corresponding to the imaginary frequencies and applying the condition that these modes connected the corresponding reactant and product complexes with the transition states properly. The gas phase calculations were carried out using the Windows versions of the Gaussian98 (G98W)60 and Gaussian03 (G03W)61 suites of programs, while for all the solvation calculations in different solvents using the IEF-PCM, the G98W suite of programs was employed. For visualization of the optimized structures and vibrational modes, the GaussView program was employed.62
3. RESULTS AND DISCUSSION 3.1. Structure and Stability. Relative Gibbs free energies of two conformers of dAdo called target and typical dAdo (with respect to that of the former) and those of two conformers of glutamic acid (GluI, GluII) (with respect to that of GluI) obtained at the BHandHLYP/AUG-cc-pVDZ and MP2/AUGcc-pVDZ levels of theory employing the gas-phase geometries optimized at the B3LYP/6-31G(d,p) level are presented in Table 1. This table contains relative Gibbs free energies obtained at the BHandHLYP/AUG-cc-pVDZ level in two different solvents (water and chlorobenzene) and gas-phase Gibbs free
energies obtained at the other two levels. The optimized structures of these molecules and their relative Gibbs free energies obtained at the MP2/AUG-cc-pVDZ level are presented in Figure 1. In view of the fact that the MP2 method treats electron correlation more accurately than DFT and as the dielectric constants of chlorobenzene and dichloroethane are moderate (much less than that of water), gas-phase MP2/AUGcc-pVDZ level calculations would be expected to indicate trends of results reliably. The target and typical dAdo differ with respect to the dihedral angle C50 C40 C30 O30 which is intimately linked to the DNA backbone conformation. In a previous crystallographic study,63-65 values of the dihedral angle C50 C40 C30 O30 for target and typical dAdo were found to be 152° ( 6° and 130° ( 18°, respectively. Our optimized values of the dihedral angle C50 C40 C30 O30 for target and typical dAdo were found to be 153° and 149°, respectively. While the optimized value of the dihedral angle C50 C40 C30 O30 for target dAdo is close to the experimental one, the optimized value corresponding to typical dAdo is seemingly quite different from the experimental value. However, if we consider the possible error limit of the experimental value for typical dAdo, the difference between our optimized and experimental values of this dihedral angle also becomes acceptable. The calculated Gibbs free energies in the gas phase at the MP2/AUG-cc-pVDZ level of theory show that the target dAdo is more stable than the typical dAdo by 1.6 kcal/mol (Table 1, Figures 1a,b). Odai et al.66 have found four distinct conformers of zwitterionic glutamic acid. We considered the canonical (nonzwitterionic) form of glutamic acid in this study as in the enzyme MutY, and glutamic acid would participate in the nonzwitterionic form. The relative Gibbs free energies of the two most stable of these nonzwitterionic conformers denoted by GluI and GluII (Table 1, Figure 1c,d) were found in the present study at the MP2/AUG-cc-pVDZ level of theory in the gas phase to be 0 and 1.0 kcal/mol, respectively. The present study of the reaction in question was performed considering the more stable of the two conformers of glutamic acid (GluI) as discussed below. The optimized structures of the reactant complex of target dAdo with each of the two conformers of glutamic acid (GluI and GluII) along with two water molecules, denoted as RC1 and RC2, respectively, at the B3LYP/6-31G(d,p) level of theory in the gas phase are presented in Figure 2. A previous X-ray crystallographic33 study has also shown the presence of two water molecules placed in catalytically relevant positions in the active site of the system, similar to what is obtained by us (Figure 2). One of these water molecules is hydrogen bonded to the adenine N7 atom, while the other is positioned near the C10 atom of the sugar moiety. We would refer to the water molecule hydrogen bonded to the adenine N7 site as the first water molecule, while that positioned near the C10 atom of the sugar moiety would be referred to as the second water molecule in our discussion. Values of some important geometrical parameters of RC1 and RC2 are given in Figure 2. According to the Gibbs free energies obtained at the BHandHLYP/AUG-ccpVDZ level of theory, RC2 is less stable than RC1 in aqueous media by 0.4 kcal/mol, while in chlorobenzene, at the BHandHLYP/AUG-cc-pVDZ level of theory, this difference is found to be 0.3 kcal/mol and at the MP2/AUG-cc-pVDZ level of theory in the gas phase is found to be 0.5 kcal/mol (Figure 2). The complexes RC1 and RC2 are stabilized by four hydrogen bonds each: first a strong hydrogen bond between the N7 atom of target dAdo and the H10 atom of the first water molecule, second 3202
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Figure 2. Structures of reactant complexes RC1 and RC2 of dAdo with (a) GluI and two water molecules and (b) GluII with two water molecules. The relative Gibbs free energies (kcal/mol) of RC1 and RC2 obtained at the MP2/AUG-cc-pVDZ level of theory in the gas phase using the optimized geometry at the B3LYP/6-31G(d,p) level in the gas phase are given with respect to that of RC1. Atomic numbering scheme and some optimized geometrical parameters (Å, degree) obtained at the B3LYP/6-31G(d,p) level in the gas phase are given.
between the H11 atom of glutamic acid and the O10 atom of the first water molecule, third between the O13 atom of glutamic acid and the H20 atom of the second water molecule, and fourth between the H6 atom of the amino group of target dAdo and the O10 atom of the first water molecule. The N7H10, H11O10, O13H20, and H6O10 hydrogen bonding distances in RC1 and RC2 are shown in Figure 2. We find that the first three of these hydrogen bonds are strong, while the fourth one is weak in each case. As RC1 is more stable than RC2, reactions between target dAdo and glutamic acid were considered to involve the former. However, in view of a small total energy difference between RC1 and RC2, both these complexes would exist, of course in different abundances, and would be involved in similar reactions. 3.2. Three-Step Mechanism of Glycosidic Bond Cleavage of dAdo. We studied the mechanism of excision of the glycosidic bond of dAdo starting with the reactant complex RC1. In these calculations, we primarily investigated the energetics of the threestep mechanism suggested by McCann and Berti.28,67 Details of the reaction involving the reactant complex RC1, transition states TS1, TS2, and TS3, intermediate complexes IC1 and IC2, and the product complex PC are shown in Figure 3. Gibbs
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free energy changes corresponding to the barrier and released energies (kcal/mol) obtained at the MP2/AUG-cc-pVDZ level of theory in the gas phase as discussed below are given in Figure 3. The net CHelpG charges (in the unit of magnitude of electronic charge) obtained at the MP2/AUG-cc-pVDZ level of theory in the gas phase using the geometries optimized at the B3LYP/631G(d,p) level of theory in the gas phase, along with certain optimized geometrical parameters, are also shown in Figure 3. The dotted arrow in this figure shows formation of the PC directly from IC1 as discussed in the next subsection. The ZPE-corrected barrier and released energies pertaining to different steps of the reaction under study and the corresponding Gibbs free energy changes obtained at the B3LYP/6-31G(d,p), B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-cc-pVDZ, and MP2/AUG-cc-pVDZ levels of theory in gas phase are presented in Table 2. This table also contains the ZPE-corrected barrier and released energies and the corresponding Gibbs free energy changes obtained in three different solvents, i.e., chlorobenzene, dichloroethane, and water, which are characterized by different dielectric constants. Due to convergence failure, we could not find the ZPE-corrected barrier and released energies, and the corresponding Gibbs free energy changes in aqueous media at the MP2/AUG-cc-pVDZ level of theory. Further, the gas-phase results obtained at the B3LYP/AUG-cc-pVDZ and BHandHLYP/AUG-cc-pVDZ levels are broadly similar to the corresponding results in chlorobenzene and dichloroethane, and the same would be expected for the MP2/AUG-cc-pVDZ level results also. The following results were obtained by geometry optimization calculations in the gas phase at the B3LYP/6-31G(d,p) level. At the first step of the reaction, the N7 site of target dAdo gets protonated as the H10 proton is detached from the first water molecule and gets associated with it. At the same time, when the H10 proton is detached from the first water molecule, the H11 proton gets dissociated from glutamic acid to get attached to the OH group of the first water molecule. Thus, the intermediate complex IC1 is formed (Figure 3). The N7H10 distance at TS1 is 1.175 Å which is appreciably larger than the normal NH bond length. At TS1, the CHelpG charges associated with adenine, sugar, and glutamic acid moieties were found at the MP2/AUGcc-pVDZ level in the gas phase to be 0.50, 0.24, and -0.62, respectively. At IC1, the CHelpG charges at the protonated adenine, sugar, and deprotonated glutamic acid moieties were found at the MP2/AUG-cc-pVDZ level in the gas phase to be 0.57, 0.24, and -0.68, respectively. The first and third of these CHelpG charges conform to the protonated and deprotonated states of adenine and glutamic acid, respectively. The Gibbs free barrier energy (ΔG1b) for this step of the reaction was found to be 8.2 kcal/mol at the MP2/AUG-cc-pVDZ level of theory in the gas phase (Table 2). Further, the values of this barrier energy obtained in the gas phase,in the solvents chlorobenzene and dichloroethane at the other levels of theory are also close to this value or are not too different from it. In an experimental work,33 the barrier energy involved in the protonation of N7 of adenine was reported to be 7 kcal/mol. Thus, our calculated barrier energies corresponding to TS1 are in a good agreement with the experimental value.33 In going from the gas phase to aqueous media at the B3LYP/ AUG-cc-pVDZ and the BHandHLYP/AUG-cc-pVDZ levels of theory, IC1 gets shifted upward in energy significantly with respect to TS1 (by ∼9 kcal/mol), while in each of the other two solvents, it is shifted downward slightly. Consequently, in aqueous media, IC1 would lie appreciably higher than TS1 3203
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Figure 3. Reaction mechanism of cleavage of the glycosidic bond of dAdo by glutamic acid and two water molecules. Gibbs free energy changes corresponding to the barrier (ΔGib) and released (ΔGir) (i = 1-3) energies (kcal/mol) and net CHelpG charges (in brackets) obtained at the MP2/ AUG-cc-pVDZ level of theory in the gas phase and certain optimized geometrical parameters (Å, degree) obtained at the B3LYP/6-31G(d,p) level of theory in the gas phase are given. The Gibbs free energies are not to scale. The dotted arrow shows formation of PC directly from IC1 according to the new proposed mechanism.
(by ∼6.7 kcal/mol). Although this treatment of solvent effect is approximate as the gas phase geometry was used in the solvation calculations, in view of the large upward shift of IC1 in energy with respect to TS1, it appears that the reaction would not occur in aqueous media while it can occur in the other two solvents that would approximately represent the environment of MutY complexed with target dAdo. At the second step of the reaction, the glycosidic bond cleavage occurs, the N9 site of the adenine moiety of dAdo getting detached from the C10 site of the sugar moiety. The intermediate complex IC2 involving an oxacarbenium cation of the sugar moiety is thus formed. At the transition state TS2, at the
MP2/AUG-cc-pVDZ level of theory in the gas phase, the CHelpG charges at the adenine, sugar, and deprotonated glutamic acid moieties were found to be -0.0, 0.79, and -0.81, respectively, while the corresponding charges at the intermediate complex IC2 were found to be -0.04, 0.82, and -0.72, respectively. The latter two charges at each of TS2 and IC2 conform to the cationic and anionic states of the sugar and glutamic acid moieties, respectively. The Gibbs free barrier energy (ΔG2b) associated with this step of the reaction is found to be 31.9 kcal/ mol at the MP2/AUG-cc-pVDZ level in the gas phase (Table 2). Convergence was not achieved in the solvation calculations for TS2 in chlorobenzene at the B3LYP/AUG-cc-PVDZ and 3204
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Table 2. ZPE-Corrected Barrier (ΔEib) and Released (ΔEir) Energies and the Corresponding Gibbs Free Energy Changes (ΔGib and ΔGir, Respectively) (kcal/mol) (i = 1-3) Involved in the Cleavage of the Glycosidic Bond of 20 -Deoxyadenosine by Glutamic Acid and Two Water Molecules Obtained at Different Levels of Theory Level of Theoryb barrier, released B3LYP/AUG-cc-pVDZ
energies, and corresponding a
Gibbs free energy changes
B3LYP/6-31G (d,p) ε = 1
BHandHLYP/AUG-cc-pVDZ
c
ε = 5.6
ε = 10.4
ε = 78.4
d
e
f
ε=1
c
ε = 5.6d ε = 10.4e ε = 78.4f
MP2/AUG-cc-pVDZc
ΔE1b
3.9
3.9
4.8
5.1
2.1
7.1
7.8
7.7
5.3
ΔG1b
7.3
7.3
8.2
8.5
5.5
10.5
11.2
11.1
8.7
8.2
ΔE1r
1.2
-0.1
-0.3
-0.5
9.1
-1.6
-1.9
-2.2
7.7
-0.8
ΔG1r
-0.4
-1.7
-1.9
-2.1
7.5
-3.2
-3.5
-3.8
6.1
-2.4
ΔE2b ΔG2b
28.0 28.5
30.0 30.5
28.7 29.2
19.8 20.3
33.0 33.5
31.9 32.4
22.6 23.1
31.4 31.9
4.8
ΔE2r
-3.4
-2.9
-1.5
-1.3
-3.7
-1.7
-1.5
-4.6
ΔG2r
-2.7
-2.2
-0.8
-0.6
-3.0
-1.0
-0.8
-3.9
ΔE3b
-0.1
0.4
1.1
-0.3
0.3
1.2
1.4
0.2
0.7
1.4
ΔG3b
0.9
1.4
2.1
0.7
1.3
2.2
2.4
1.2
1.7
2.4
ΔE3r
-32.8
-28.5
-27.4
-27.6
-23.0
-34.2
-32.9
-33.3
-32.3
-36.7
ΔG3r
-32.9
-28.6
-27.5
-27.7
-23.1
-34.3
-33.0
-33.4
-32.4
-36.8
a
See Figure 3 for a definition of the energies. b Single-point energy calculations were performed at the B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-ccpVDZ, and MP2/AUG-cc-pVDZ levels in the gas phase employing the gas-phase optimized geometries at the B3LYP/6-31G** level. For solvation, single-point energy calculations were performed using the IEF-PCM. c ε = 1.0 (gas phase). d ε = 5.6 (chlorobenzene). e ε = 10.4 (dichloroethane). f ε = 78.4 (water).
BHandHLYP/AUG-cc-pVDZ levels of theory. At the other levels of theory, in the gas phase and the solvents dichloroethane and water, the calculated barrier energies corresponding to TS2 lie in the range 19.8-33.5 kcal/mol (Table 2). These values of the barrier energy are quite large on the basis of which it appears that the reaction would not occur efficiently. At the third step of the reaction (Figure 3), the second water molecule gets dissociated, the OH group of which gets attached to the oxacarbenium cation, while the proton gets associated with the anion of glutamic acid. The Gibbs free barrier energy (ΔG3b) involved at this step of the reaction is found to be quite low, i.e., 2.4 kcal/mol at the MP2/ AUG-cc-pVDZ level of theory in the gas phase, and the corresponding calculated barrier energies at the other levels of theory are all similar (Table 2). It can be ascribed to a high reactivity of the oxacarbenium cation as discussed elsewhere.68-73 3.3. New Proposed Two-Step Reaction Mechanism. The results presented above pose a serious difficulty. That is, it appears difficult to understand how the reaction would occur as the second barrier energy is quite high. Reactions catalyzed by enzymes (in the present case, the active site of the enzyme MutY is represented by glutamic acid and two water molecules) are expected to involve low barrier energies. This difficulty can be resolved on the basis of our calculations, considering an alternative mechanism to what is discussed above, as follows. The net Gibbs free energy changes which can be obtained by the sums ΔG1b þ ΔG2b þ ΔG3b þ ΔG1r þ ΔG2r þ ΔG3r of the Gibbs free barrier and released energies calculated at the different levels of density functional theory employed here in the gas phase, dichloroethane, and water were found to lie between 0.7 and 10.9 kcal/mol, while the corresponding sum at the MP2/ AUG-cc-pVDZ level in the gas phase was found to be -0.7 kcal/ mol (Table 2). Further, these sums were found to be more (positive) in the solvent media than in the gas phase. These results suggest that the overall reaction starting from the RC1 and ending in the PC in the biological media would be endothermic
by a few kilocalories/mole of energy. However, this endothermicity arises mainly due to the first step (corresponding to TS1) of the reaction as the sums ΔG2b þ ΔG3b þ ΔG2r þ ΔG3r of energies in the gas phase and the different solvents at the various levels of theory were found to be mostly negative and only in two cases mildly positive (Table 2). In particular, in aqueous media, these sums at the B3LYP/AUG-cc-pVDZ and BHandHLYP/ AUG-cc-pVDZ levels of theory were found to be -2.1 and -8.4 kcal/mol, respectively. These results show that in biological media PC would lie lower than IC1. It appears that a new reaction mechanism would operate as follows. The second and third steps of the reaction may not occur separately; instead, it appears likely that these two steps would get combined and occur as a single step. It would imply that dissociation of the water molecule and attack of the OH- moiety (as discussed later) at the C10 site of the sugar moiety would occur at the second step itself. We note that PC lies lower than IC1 in the gas phase at the B3LYP/6-31G(d,p) level by -6.2 kcal/mol (Table 2). Therefore, we tried to locate the possible transition state between IC1 and PC allowing the water molecule located near the sugar moiety to get dissociated and the OH- moiety to attack the C10 site. This calculation resulted in formation of the PC, but no transition state could be located even after repeated attempts considering different possible orientations of the various species involved (Figure 3). Thus, it appears that starting from IC1, PC would be formed barrierlessly. If a transition state is flat, it may not be possible to locate it. It appears to be true in the present case. Certain reactions have been found to be barrierless earlier also.6 It has been reported earlier52 that the reaction step associated with the neucleophilic attack of OHat the C10 site is barrierless. To obtain further information about the nature of the potential energy surface between IC1 and PC, additional calculations were performed. These results are presented and discussed in detail in the Supporting Information. These calculations were 3205
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The Journal of Physical Chemistry B started with six interpolated points with respect to certain distances lying between IC1 and PC that would be involved in the reaction under consideration. Subsequently, energy minimization to a limited extent (10 cycles) was performed at the B3LYP/6-31G(d,p) level of theory in the gas phase to relax the possible undue constraints at each of the interpolated points. Vibrational frequency calculations at each of the points obtained by limited energy minimization yielded more than one imaginary frequency. The atomic displacements corresponding to one of the imaginary frequencies at each of the points clearly showed dissociation of the glycosidic bond C10 N9, while in the other three cases, atomic displacements involving bonds of the water molecule and the glutamic acid moiety were prominent. Starting from each of five of the points thus obtained, dissociating the water molecule located near C10 , placing the OH group near C10 thereby facilitating attack of the former at the latter in accordance with the findings of a previous study,74 calculations were performed to search the possible transition state. None of these calculations yielded a transition state, and it was found that during the process of transition state search the interatomic distances usually changed in the direction from IC1 to PC. An examination of the variation of total energy and rms gradient on the potential energy surface with respect to optimization cycles during the transition state search started from one of the points lying in the middle region suggested that there may be shallow wiggles between IC1 and PC on the potential energy surface, but no convincing indication supporting the occurrence of a barrier seemed to exist. Thus it appears that the surface in the transition state region between IC1 and PC would be flat and would correspond to a vanishingly small barrier energy. Thus the reaction step between IC1 and PC would be barrierless or nearly barrierless. An examination of the net CHelpG charges associated with the different components of IC1 and PC shows that adenine and sugar moiety together would gain negative charge (sum of the two components ∼ -0.67), while the glutamic acid moiety would lose the same significantly (∼0.46) at the second step of the new proposed reaction mechanism. In going from IC1 to PC, charges are also significantly rearranged at the first water molecule (Figure 3). These net charges suggest that the water molecule located near the sugar moiety would get dissociated into OH- and Hþ: the former group would attack the C10 site of the sugar moiety, while the latter would get attached to the glutamic acid. Thus the water molecule located near the sugar moiety would participate in the reaction under consideration as OH- and Hþ ions and not as OH and H radicals. We also addressed the question whether the entire reaction starting from the RC1 and ending in the PC could be completed in a single step. Calculations performed with regard to this question showed that the first step (Figure 3) always occurred independently and led to the formation of IC1 from RC1. Thus, formation of the PC, starting from IC1, must be treated as the second and final step. Therefore, it appears most likely that the reaction in question would be a two-step one, though there is only one significant energy barrier involved in it. This study shows that the MutY-catalyzed repair of DNA involving dissociation of the glycosidic bond would occur much more rapidly than what is indicated by the three-step mechanism proposed in the previous study.28 The present analysis, on the whole, supports the two-step mechanism for MutY-catalyzed cleavage of the glycosidic bond of 20 -deoxyadenosine in preference to the threestep mechanism.28
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4. CONCLUSIONS The present study leads us to the following important conclusions: 1. Glutamic acid and two complexed water molecules serve as a satisfactory model for the active site of MutY which catalyzes cleavage of the glycosidic bond of 20 -deoxyadenosine. 2. We have proposed here a two-step mechanism for the MutY-catalyzed cleavage of the glycosidic bond of 20 deoxyadenosine. It differs from the three-step mechanism proposed earlier on the basis of experimental TS analysis. The first step and the final product are common between the two mechanisms. 3. The second step of the reaction mechanism proposed earlier was found to be associated with a prohibitively high calculated barrier energy. In the two-step mechanism proposed here, this step is obviated as the product complex (PC) is formed directly in a nearly barrierless manner from the intermediate complex (IC1) formed at the first reaction step. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional details. This material is available free of charge via the Internet at http://pubs.acs. org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Notes †
Deceased.
’ ACKNOWLEDGMENT The authors are thankful to the Council of Scientific and Industrial Research (New Delhi) and the University Grants Commission (New Delhi) for financial support. ’ REFERENCES (1) Burrows, C. J.; Muller, J. G. Chem. Rev. 1998, 98, 1109. (2) Halliwell, B. Mutat. Res. 1999, 443, 37. (3) O’Brien, P. Chem. Rev. 2006, 106, 302. (4) Simons, J. Acc. Chem. Res. 2006, 39, 772. (5) Wyatt, M. D.; Pittman, D. L. Chem. Res. Toxicol. 2006, 19, 1580. (6) Jena, N. R.; Mishra, P. C. J. Phys. Chem. B 2005, 109, 14205. (7) Shukla, P. K.; Mishra, P. C. J. Phys. Chem. B 2008, 112, 4779. (8) Shukla, M. K.; Mishra, P. C. J. Mol. Struct. 1996, 377, 247. (9) Srivastava, S. K.; Mishra, P. C. Int. J. Quantum Chem. 1979, 16, 1051. (10) Venkateswarlu, D.; Leszczynski, J. J. Comput.-Aided Mol. Des. 1998, 12, 373. (11) Agnihotri, N.; Mishra, P. C. J. Phys. Chem. B 2009, 113, 3129. (12) Neeley, W. L.; Essigmann, J. M. Chem. Res. Toxicol. 2006, 19, 491. (13) Bignami, M.; O’ Driscoll, M.; Aquilina, G.; Karran, P. Mutat. Res. 2000, 462, 71. (14) Mishra, S. K.; Mishra, P. C. J. Comput. Chem. 2002, 23, 530. (15) Tiwari, S.; Mishra, P. C. J. Mol. Model. 2011, 17, 59. (16) Shukla, M. K.; Mishra, P. C. Spectrochim. Acta 1995, 51A, 831. (17) Pegg, A. E.; Fang, Q.; Loktionova, N. A. DNA Repair 2007, 6, 1071. 3206
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