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Theoretical study on the ring-opening hydrolysis reaction of cAMP
Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152 www.elsevier.com/locate/theochem
Theoretical study on the ring-opening hydrolysis reaction of cAMP Aihua Zhang, Kun Liu, Caixia Wang, Siyu Ma, Zonghe Li* Department of Chemistry, Beijing Normal University, Beijing 100875, China Received 20 November 2004; revised 27 December 2004; accepted 27 December 2004
Abstract The ring-opening hydrolysis reaction mechanism of cyclic 3 0 , 5 0 -adenosine monophosphate (cAMP) have been theoretically investigated at the B3LYP/6-31G** level. It is found that each ring-opening hydrolysis reaction of cAMP takes place via a four-membered transition state. For the reactions, H2O has two attacking sites (a and b). At each site, H2O has two attacking objects. One is the P atom, the other is the bond P–O, and they are competitive each other. The potential energy surface of reaction 4 is the lowest. The computations strongly support the experimental result that the nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP is the most advantageous hydrolysis reaction path. Our present calculations have rationalized and verified all the possible reaction channels. q 2005 Elsevier B.V. All rights reserved. Keywords: cAMP; B3LYP; 5 0 -AMP; Ring-opening hydrolysis
1. Introduction Cyclic 3 0 , 5 0 -adenosine monophosphate (cAMP) is a key molecule in living organisms, which was discovered in 1958 by Sutherland and Rall [1]. It is known as the intracellular second messenger and plays an essential role in vision, muscle contraction, neurotransmission, exocytosis, cell growth and differentiation [2]. As a member of a series of nucleotides, the structure of cAMP is relative simple, so it is the entrance of theoretical study on a series of nucleotides. At the same time, the thermochemistry reaction mechanism of cAMP has received much attention and theoretical viewpoints, especially its ring-opening hydrolysis reaction. The extensive studies of phosphoester hydrolysis reactions by Karplus [3–6] and by Warshel [7,8] indeed provide such a justification for the use of a small basis set in this work. The recent reports of cyclic phosphate ester hydrolysis by Chen [9] and by Holmes [10] provide a certain help for the theoretical study on the ring-opening hydrolysis reaction mechanism of cAMP. Previous experiments have demonstrated that the ring-opening hydrolysis pathways include both enzymatic [11–13] and nonenzymatic [14] ester
* Corresponding author. Tel.: C86 1062207404; fax: C86 1062207404. E-mail address: [email protected] (Z. Li). 0166-1280/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2004.12.034
hydrolysis. Enzymatic hydrolysis leads to 5 0 -AMP [15–18], whereas nonenzymatic hydrolysis yields 3 0 -AMP [14]. To our best knowledge, no detailed theoretical study has been performed on the hydrolysis reaction mechanism of cAMP using B3LYP/6-31G** method. In this paper, we have theoretically investigated on the ring-opening hydrolysis reaction pathways.
2. Methods of calculation For all reactions under consideration in this study, we have first employed B3LYP method [19–22] of density functional theory (DFT) with the standard 3-21G** basis set to search for and optimized all possible geometries of transition states as well as reactants, complexes, intermediates and products. The geometries obtained at the B3LYP/ 3-21G** level are then refined at the B3LYP/6-31G** level. The harmonic frequency calculations are performed at the same level to verify whether they are minima with all real frequencies or transition states with only one imaginary frequency. For the key reaction paths, intrinsic reaction coordinates (IRC) [23] have been traced to confirm the TS connecting with the corresponding two minima. All calculations are performed with the GAUSSIAN-98 [24] package on a P4-2G computer.
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A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
3. Results and discussion 3.1. The ring-opening hydrolysis reaction mechanism of cAMP The ring-opening hydrolysis reaction mechanism of cAMP is investigated in the gas phase and four possible hydrolysis pathways in this reaction are discussed. The geometries of all
stationary points are fully optimized using B3LYP/6-31G** method, followed by harmonic frequency calculations to confirm that the reactants, products and intermediates with all real frequencies and transition states with only one imaginary frequency. The optimized structures are displayed in Fig. 1 and some of geometrical parameters are listed in it. The hydrolysis reaction of cAMP is multi-channel. Four reaction channels are got using quantum chemistry
Fig. 1. Geometries of the reactants, transition states, and intermediates optimized at the B3LYP/6-31G** level for the pathways associated with reactions.
A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
method:
151
Table 1 The total energies (a.u.), zero-point energies (a.u.), relative energies (kcal/mol) and vibrational frequencies (cmK1) in the gas phase (B3LYP/631G**)
cAMP C H2 O/ TS1/ 5 0 AMP
(1)
cAMP C H2 O/ TS21/ IM2/ TS22/ 5 0 AMP
(2)
Species
Total energies
Zero point energies
Relative energies
cAMP C H2 O/ TS3/ 3 0 AMP
(3)
cAMP C H2 O/ TS41/ IM4/ TS42/ 3 0 AMP
(4)
cAMPCH2O 5 0 -AMP 3 0 -AMP IM2 IM4 TS1 TS21 TS22 TS3 TS41 TS42
K1531.251804 K1531.287253 K1531.291317 K1531.253861 K1531.261018 K1531.200979 K1531.220044 K1531.233867 K1531.205597 K1531.224233 K1531.231174
0.273060 0.277224 0.277520 0.278820 0.279223 0.275340 0.274469 0.273947 0.275731 0.274737 0.273811
0.0 K17.9 K20.3 4.1 K0.2 35.1 22.6 13.6 32.4 20.1 15.2
In the path 1, the ring-opening hydrolysis reaction of cAMP starts with the direct interaction between the reactants H2O and the bond P1–O33 of a site. Then 5 0 -AMP is formed via transition state TS1 that is a 4-membered structure. Our reaction coordinate calculations indicate that this reaction is associated with O35 of the water attacking at P1 atom of cAMP while H36 transfers from O35 of the water to O33 of cAMP. In the process of reaction, the critical bond length of P1–O33 increases ˚ from 1.746 A ˚ (reactant) to 2.016 A ˚ (TS1), while that 0.27 A ˚ ˚ ) to for O35–P1 decreases 0.308 A from reactant (2.263 A ˚ TS1 (1.955 A). At the same time, the critical bond length of ˚ O35–H36 and H36–O33 changes as follows: 0.960 A ˚ ˚ ˚ (reactant)/1.339 A (TS1), 1.800 A (reactant)/1.098 A (TS1), respectively. Because while two critical bond lengths of P1–O33 and O35–H36 increase, another two critical bond lengths of O35–P1and H36–O33 decrease, path 1 belongs to associative ring-opening hydrolysis reaction. The IRC calculation in one direction goes to the reactant cAMP and H2O. The IRC calculation in another direction goes to the product 5 0 -AMP without going through a pentacoordinated phosphorus intermediate. Path 2 has been found to be associated with a two-step process. The first step is that water molecule from b site of cAMP attacks at P atom. The formation of a pentacoordinated phosphorus intermediate (IM2) is created via TS21. This step is associated with O35 of the water attacking at P1 atom of cAMP while a proton (H36) transfers from the water oxygen (O35) to the phosphoryl oxygen (O2). The second step is the decomposition of IM2 to 5 0 -AMP. 5 0 -AMP is formed via transition state TS22. In this step, the axial ester oxygen (O33) gradually leaves the P1 atom while a proton (H34) gradually transfers from the equatorial hydroxyl oxygen (O3) to the leaving oxygen (O33). TS21 is a 4-membered structure, with O35–H36 and H36–O2 bonds ˚ , respectively, TS22 is also a being about 1.213, 1.249 A 4-membered structure. In the process of reaction, H36 of the water transfers to O2 of the cAMP, leading to IM2 and afterwards H34 of IM2 moves towards its O33. So path 2 belongs to dissociative H shuttling hydrolysis reaction. The difference between the path 3 and path 1 is that water molecule from b site of cAMP interacts with the bond P1–O4. Then 3 0 -AMP is created via transition state TS3. The difference between the path 4 and path 2 is that water molecule from a site of cAMP attacks at P atom and the intermediate IM4 is formed via TS41. In the process of
Frequencies
K606.7 K1312.9 K871.8 K582.6 K1303.3 K1142.3
reaction, intermediate IM2 and IM4 are isomeric. The torsion of OH group happens between them. Then IM4 may decompose to the product 3 0 -AMP via TS42. 3.2. The change in potential energy The energetic data for all the critical points in the reaction (1)–(4) are calculated and summarized in Table 1 by taking the energy of reactant cAMP and H2O as zero. The potential energy profile for the ring-opening hydrolysis reaction of cAMP to 5 0 -AMP and 3 0 -AMP is depicted in Fig. 2 and four kinds of reaction mechanisms are revealed. From Fig. 2 one can see that path 1 and path 3 proceed in synchronous way. From cAMPCH2O to 5 0 -AMP, it needs to overcome 35.1 kcal/mol energy barrier in path 1, and the energy barrier from cAMPCH2O to 3 0 -AMP is 32.4 kcal/mol in path 3. And the energy of TS3 is lower 2.7 kcal/mol than that of TS1. Path 2 and path 4 have been found to be associated with a two-step process. In the reaction
Fig. 2. Schematic potential energy surfaces of hydrolysis reaction mechanism.
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A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
Table 2 Calculated energy barriers (DE) and free energy barriers (DG) in (kcal/mol) using B3LYP/6-31G** method Reaction Reaction (1) cAMPCH2O/TS1 Reaction (2) CAMPCH2O/TS21 IM2/TS22 Reaction (3) cAMPCH2O/TS3 Reaction (4) cAMPCH2O/TS41 IM4/TS42
Gas phase DE
DG
35.1
45.4
22.6 9.5
32.6 10.6
32.4
42.6
20.1 15.4
30.4 15.5
path 2, IM2 may occur via TS21 over energy barrier of 22.6 kcal/mol. Then the energy of 9.5 kcal/mol is needed to hydrolysis product 5 0 -AMP via TS22. In the reaction path 4, the conversion step (cAMPCH 2O/TS41/IM4) is endothermic by 20.1 kcal/mol. Then IM4 may directly dissociate into 3 0 -AMP via TS42 with a barrier of 15.4 kcal/ mol. So the first step is always rate-controlling for each reaction pathway, and comparison of the hydrolysis rate between different reaction pathways should focus on the freeenergy barriers for the first step. The calculated free-energy barriers in Table 2 predict that for the four reactions in the gas phase reaction 4 is the fastest because the free-energy barrier (30.4 kcal/mol) for its rate-controlling step is the lowest and reaction 1 is the slowest because the free-energy barrier (45.4 kcal/mol) is highest. Therefore, the reaction leading to 5 0 -AMP is in comparative with those leading to 3 0 -AMP, which accounts for the experimental fact that nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and the in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP (path 4) is the most advantageous hydrolysis reaction path.
4. Conclusion The ring-opening hydrolysis reaction mechanism of cAMP to 5 0 -AMP and 3 0 -AMP have been theoretically investigated at the B3LYP/6-31G** level. The results show that ring-opening hydrolysis reaction of cAMP may proceed in synchronous way or in stepwise way. The reaction coordinate calculations reveal four fundamental reaction pathways for cAMP hydrolysis: (1) attack of a water molecule from a site of cAMP at the P1–O33 bond; (2) direct attack of a water molecule from b site of cAMP at the phosphorus center (in-line nonenzymatic hydrolysis); (3) attack of a water molecule from b site of cAMP at the P1–O4 bond; and (4) direct attack of a water molecule from a site of cAMP at the phosphorus center (in-line nonenzymatic hydrolysis). Reaction pathway 1 and Reaction
pathway 3 are actually a single-step associative reaction without going through a penta-coordinated phosphorus intermediate. Path 2 and path 4 are both a two-step process with a single penta-coordinated phosphorus intermediate structure. The calculated energetic results indicate that the first step of the reaction is always the rate-controlling step for all the reaction pathways. For the reaction in the gas phase, the calculated free-energy barrier for path 1 is the highest, and that for the rate-controlling step of path 4 is the lowest. Both a single-step hydrolysis reaction and a two-step process have demonstrated the experimental fact that nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and the in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP (path 4) is the most advantageous hydrolysis reaction path.
References [1] E.W. Sutherland, T.W. Rall, J. Biol. Chem. 232 (1958) 1065. [2] S.M. Callahan, N.W. Cornell, P.V. Dunlap, J. Biol. Chem. 270 (1995) 17627. [3] A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 115 (1993) 5316. [4] A. Dejaegere, X.L. Liang, M. Karplus, J. Chem. Soc. Faraday Trans. 90 (1994) 1763. [5] X. Lopez, A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 123 (2001) 11755. [6] X. Lopez, M. Schaefer, A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 124 (2002) 5010. [7] J. Florian, A. Warshel, J. Am. Chem. Soc. 119 (1997) 5473. [8] J. Aqvist, K. Kolmodin, J. Florian, A. Warshel, Chem. Biol. 6 (1999) R71. [9] X. Chen, C.G. Zhan, J. Phys. Chem. A 108 (2004) 3789–3797. [10] R.R. Holmes, J.A. Deiters, Inorg. Chem. 33 (1994) 3235–3238. [11] P.J.J.M. Van Ool, H.M. Buck, Recl. Traw. Chim. Pays Bas 100 (1981) 79. [12] J.H. Yu, A.M. Arif, W.G. Bentrude, J. Am. Chem. Soc. 112 (1990) 7451. [13] J.H. Yu, A.E. Sopchik, A.M. Arif, W.G. Bentrude, J. Org. Chem. 55 (1990) 3444. [14] S. Mehdi, J.A. Coderre, J.A. Gerlt, Tetrahedron 39 (1983) 3483. [15] P.M.J. Bburgers, F. Eckstein, D.H. Hunneman, J. Baraniak, R.W. Kinas, K. Lesia, W.J. Stec, J. Biol. Chem. 254 (1979) 9959. [16] J.A. Coderre, S. Mehdi, J.A. Gerlt, J. Am. Chem. Soc. 103 (1981) 1872. [17] P.M. Cullis, R.L. Jarvest, G. Lowe, B.V.L. Potter, J. Chem. Soc. Chem. Commun. (1981) 245. [18] R.L. Jarvest, G. Lowe, J. Baraniak, W.J. Stec, Biochem. J. 203 (1982) 461. [19] A.D. Becke, Phys. Rev. 38 (1988) 3098. [20] C. Lee, W. Yang, R.G. Parr, Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785. [21] B. Miehlich, A. Savin, H. Stoll, et al., Chem. Phys. Lett. 157 (1989) 200. [22] A.D. Becke, Density-functional thermochemistry. III The role of exact exchange, J. Chem. Phys. 98 (1993) 5648. [23] (a) C. Gonzalez, H.B. Schlegel, J. Chem. Phys. 90 (1989) 2154; (b) C. Gonzalez, H.B. Schlegel, J. Phys. Chem. 94 (1990) 5523. [24] M.J. Frisch, G.W. Trucks, H.B. Schlegel, et al. GAUSSIAN-98, Gaussian, Inc., Pittsburgh PA, 1998.
Theoretical study on the ring-opening hydrolysis reaction of cAMP Aihua Zhang, Kun Liu, Caixia Wang, Siyu Ma, Zonghe Li* Department of Chemistry, Beijing Normal University, Beijing 100875, China Received 20 November 2004; revised 27 December 2004; accepted 27 December 2004
Abstract The ring-opening hydrolysis reaction mechanism of cyclic 3 0 , 5 0 -adenosine monophosphate (cAMP) have been theoretically investigated at the B3LYP/6-31G** level. It is found that each ring-opening hydrolysis reaction of cAMP takes place via a four-membered transition state. For the reactions, H2O has two attacking sites (a and b). At each site, H2O has two attacking objects. One is the P atom, the other is the bond P–O, and they are competitive each other. The potential energy surface of reaction 4 is the lowest. The computations strongly support the experimental result that the nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP is the most advantageous hydrolysis reaction path. Our present calculations have rationalized and verified all the possible reaction channels. q 2005 Elsevier B.V. All rights reserved. Keywords: cAMP; B3LYP; 5 0 -AMP; Ring-opening hydrolysis
1. Introduction Cyclic 3 0 , 5 0 -adenosine monophosphate (cAMP) is a key molecule in living organisms, which was discovered in 1958 by Sutherland and Rall [1]. It is known as the intracellular second messenger and plays an essential role in vision, muscle contraction, neurotransmission, exocytosis, cell growth and differentiation [2]. As a member of a series of nucleotides, the structure of cAMP is relative simple, so it is the entrance of theoretical study on a series of nucleotides. At the same time, the thermochemistry reaction mechanism of cAMP has received much attention and theoretical viewpoints, especially its ring-opening hydrolysis reaction. The extensive studies of phosphoester hydrolysis reactions by Karplus [3–6] and by Warshel [7,8] indeed provide such a justification for the use of a small basis set in this work. The recent reports of cyclic phosphate ester hydrolysis by Chen [9] and by Holmes [10] provide a certain help for the theoretical study on the ring-opening hydrolysis reaction mechanism of cAMP. Previous experiments have demonstrated that the ring-opening hydrolysis pathways include both enzymatic [11–13] and nonenzymatic [14] ester
* Corresponding author. Tel.: C86 1062207404; fax: C86 1062207404. E-mail address: [email protected] (Z. Li). 0166-1280/$ - see front matter q 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2004.12.034
hydrolysis. Enzymatic hydrolysis leads to 5 0 -AMP [15–18], whereas nonenzymatic hydrolysis yields 3 0 -AMP [14]. To our best knowledge, no detailed theoretical study has been performed on the hydrolysis reaction mechanism of cAMP using B3LYP/6-31G** method. In this paper, we have theoretically investigated on the ring-opening hydrolysis reaction pathways.
2. Methods of calculation For all reactions under consideration in this study, we have first employed B3LYP method [19–22] of density functional theory (DFT) with the standard 3-21G** basis set to search for and optimized all possible geometries of transition states as well as reactants, complexes, intermediates and products. The geometries obtained at the B3LYP/ 3-21G** level are then refined at the B3LYP/6-31G** level. The harmonic frequency calculations are performed at the same level to verify whether they are minima with all real frequencies or transition states with only one imaginary frequency. For the key reaction paths, intrinsic reaction coordinates (IRC) [23] have been traced to confirm the TS connecting with the corresponding two minima. All calculations are performed with the GAUSSIAN-98 [24] package on a P4-2G computer.
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A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
3. Results and discussion 3.1. The ring-opening hydrolysis reaction mechanism of cAMP The ring-opening hydrolysis reaction mechanism of cAMP is investigated in the gas phase and four possible hydrolysis pathways in this reaction are discussed. The geometries of all
stationary points are fully optimized using B3LYP/6-31G** method, followed by harmonic frequency calculations to confirm that the reactants, products and intermediates with all real frequencies and transition states with only one imaginary frequency. The optimized structures are displayed in Fig. 1 and some of geometrical parameters are listed in it. The hydrolysis reaction of cAMP is multi-channel. Four reaction channels are got using quantum chemistry
Fig. 1. Geometries of the reactants, transition states, and intermediates optimized at the B3LYP/6-31G** level for the pathways associated with reactions.
A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
method:
151
Table 1 The total energies (a.u.), zero-point energies (a.u.), relative energies (kcal/mol) and vibrational frequencies (cmK1) in the gas phase (B3LYP/631G**)
cAMP C H2 O/ TS1/ 5 0 AMP
(1)
cAMP C H2 O/ TS21/ IM2/ TS22/ 5 0 AMP
(2)
Species
Total energies
Zero point energies
Relative energies
cAMP C H2 O/ TS3/ 3 0 AMP
(3)
cAMP C H2 O/ TS41/ IM4/ TS42/ 3 0 AMP
(4)
cAMPCH2O 5 0 -AMP 3 0 -AMP IM2 IM4 TS1 TS21 TS22 TS3 TS41 TS42
K1531.251804 K1531.287253 K1531.291317 K1531.253861 K1531.261018 K1531.200979 K1531.220044 K1531.233867 K1531.205597 K1531.224233 K1531.231174
0.273060 0.277224 0.277520 0.278820 0.279223 0.275340 0.274469 0.273947 0.275731 0.274737 0.273811
0.0 K17.9 K20.3 4.1 K0.2 35.1 22.6 13.6 32.4 20.1 15.2
In the path 1, the ring-opening hydrolysis reaction of cAMP starts with the direct interaction between the reactants H2O and the bond P1–O33 of a site. Then 5 0 -AMP is formed via transition state TS1 that is a 4-membered structure. Our reaction coordinate calculations indicate that this reaction is associated with O35 of the water attacking at P1 atom of cAMP while H36 transfers from O35 of the water to O33 of cAMP. In the process of reaction, the critical bond length of P1–O33 increases ˚ from 1.746 A ˚ (reactant) to 2.016 A ˚ (TS1), while that 0.27 A ˚ ˚ ) to for O35–P1 decreases 0.308 A from reactant (2.263 A ˚ TS1 (1.955 A). At the same time, the critical bond length of ˚ O35–H36 and H36–O33 changes as follows: 0.960 A ˚ ˚ ˚ (reactant)/1.339 A (TS1), 1.800 A (reactant)/1.098 A (TS1), respectively. Because while two critical bond lengths of P1–O33 and O35–H36 increase, another two critical bond lengths of O35–P1and H36–O33 decrease, path 1 belongs to associative ring-opening hydrolysis reaction. The IRC calculation in one direction goes to the reactant cAMP and H2O. The IRC calculation in another direction goes to the product 5 0 -AMP without going through a pentacoordinated phosphorus intermediate. Path 2 has been found to be associated with a two-step process. The first step is that water molecule from b site of cAMP attacks at P atom. The formation of a pentacoordinated phosphorus intermediate (IM2) is created via TS21. This step is associated with O35 of the water attacking at P1 atom of cAMP while a proton (H36) transfers from the water oxygen (O35) to the phosphoryl oxygen (O2). The second step is the decomposition of IM2 to 5 0 -AMP. 5 0 -AMP is formed via transition state TS22. In this step, the axial ester oxygen (O33) gradually leaves the P1 atom while a proton (H34) gradually transfers from the equatorial hydroxyl oxygen (O3) to the leaving oxygen (O33). TS21 is a 4-membered structure, with O35–H36 and H36–O2 bonds ˚ , respectively, TS22 is also a being about 1.213, 1.249 A 4-membered structure. In the process of reaction, H36 of the water transfers to O2 of the cAMP, leading to IM2 and afterwards H34 of IM2 moves towards its O33. So path 2 belongs to dissociative H shuttling hydrolysis reaction. The difference between the path 3 and path 1 is that water molecule from b site of cAMP interacts with the bond P1–O4. Then 3 0 -AMP is created via transition state TS3. The difference between the path 4 and path 2 is that water molecule from a site of cAMP attacks at P atom and the intermediate IM4 is formed via TS41. In the process of
Frequencies
K606.7 K1312.9 K871.8 K582.6 K1303.3 K1142.3
reaction, intermediate IM2 and IM4 are isomeric. The torsion of OH group happens between them. Then IM4 may decompose to the product 3 0 -AMP via TS42. 3.2. The change in potential energy The energetic data for all the critical points in the reaction (1)–(4) are calculated and summarized in Table 1 by taking the energy of reactant cAMP and H2O as zero. The potential energy profile for the ring-opening hydrolysis reaction of cAMP to 5 0 -AMP and 3 0 -AMP is depicted in Fig. 2 and four kinds of reaction mechanisms are revealed. From Fig. 2 one can see that path 1 and path 3 proceed in synchronous way. From cAMPCH2O to 5 0 -AMP, it needs to overcome 35.1 kcal/mol energy barrier in path 1, and the energy barrier from cAMPCH2O to 3 0 -AMP is 32.4 kcal/mol in path 3. And the energy of TS3 is lower 2.7 kcal/mol than that of TS1. Path 2 and path 4 have been found to be associated with a two-step process. In the reaction
Fig. 2. Schematic potential energy surfaces of hydrolysis reaction mechanism.
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A. Zhang et al. / Journal of Molecular Structure: THEOCHEM 719 (2005) 149–152
Table 2 Calculated energy barriers (DE) and free energy barriers (DG) in (kcal/mol) using B3LYP/6-31G** method Reaction Reaction (1) cAMPCH2O/TS1 Reaction (2) CAMPCH2O/TS21 IM2/TS22 Reaction (3) cAMPCH2O/TS3 Reaction (4) cAMPCH2O/TS41 IM4/TS42
Gas phase DE
DG
35.1
45.4
22.6 9.5
32.6 10.6
32.4
42.6
20.1 15.4
30.4 15.5
path 2, IM2 may occur via TS21 over energy barrier of 22.6 kcal/mol. Then the energy of 9.5 kcal/mol is needed to hydrolysis product 5 0 -AMP via TS22. In the reaction path 4, the conversion step (cAMPCH 2O/TS41/IM4) is endothermic by 20.1 kcal/mol. Then IM4 may directly dissociate into 3 0 -AMP via TS42 with a barrier of 15.4 kcal/ mol. So the first step is always rate-controlling for each reaction pathway, and comparison of the hydrolysis rate between different reaction pathways should focus on the freeenergy barriers for the first step. The calculated free-energy barriers in Table 2 predict that for the four reactions in the gas phase reaction 4 is the fastest because the free-energy barrier (30.4 kcal/mol) for its rate-controlling step is the lowest and reaction 1 is the slowest because the free-energy barrier (45.4 kcal/mol) is highest. Therefore, the reaction leading to 5 0 -AMP is in comparative with those leading to 3 0 -AMP, which accounts for the experimental fact that nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and the in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP (path 4) is the most advantageous hydrolysis reaction path.
4. Conclusion The ring-opening hydrolysis reaction mechanism of cAMP to 5 0 -AMP and 3 0 -AMP have been theoretically investigated at the B3LYP/6-31G** level. The results show that ring-opening hydrolysis reaction of cAMP may proceed in synchronous way or in stepwise way. The reaction coordinate calculations reveal four fundamental reaction pathways for cAMP hydrolysis: (1) attack of a water molecule from a site of cAMP at the P1–O33 bond; (2) direct attack of a water molecule from b site of cAMP at the phosphorus center (in-line nonenzymatic hydrolysis); (3) attack of a water molecule from b site of cAMP at the P1–O4 bond; and (4) direct attack of a water molecule from a site of cAMP at the phosphorus center (in-line nonenzymatic hydrolysis). Reaction pathway 1 and Reaction
pathway 3 are actually a single-step associative reaction without going through a penta-coordinated phosphorus intermediate. Path 2 and path 4 are both a two-step process with a single penta-coordinated phosphorus intermediate structure. The calculated energetic results indicate that the first step of the reaction is always the rate-controlling step for all the reaction pathways. For the reaction in the gas phase, the calculated free-energy barrier for path 1 is the highest, and that for the rate-controlling step of path 4 is the lowest. Both a single-step hydrolysis reaction and a two-step process have demonstrated the experimental fact that nonenzymatic hydrolysis of cAMP yields 3 0 -AMP and the in-line nonenzymatic hydrolysis of cAMP to 3 0 -AMP (path 4) is the most advantageous hydrolysis reaction path.
References [1] E.W. Sutherland, T.W. Rall, J. Biol. Chem. 232 (1958) 1065. [2] S.M. Callahan, N.W. Cornell, P.V. Dunlap, J. Biol. Chem. 270 (1995) 17627. [3] A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 115 (1993) 5316. [4] A. Dejaegere, X.L. Liang, M. Karplus, J. Chem. Soc. Faraday Trans. 90 (1994) 1763. [5] X. Lopez, A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 123 (2001) 11755. [6] X. Lopez, M. Schaefer, A. Dejaegere, M. Karplus, J. Am. Chem. Soc. 124 (2002) 5010. [7] J. Florian, A. Warshel, J. Am. Chem. Soc. 119 (1997) 5473. [8] J. Aqvist, K. Kolmodin, J. Florian, A. Warshel, Chem. Biol. 6 (1999) R71. [9] X. Chen, C.G. Zhan, J. Phys. Chem. A 108 (2004) 3789–3797. [10] R.R. Holmes, J.A. Deiters, Inorg. Chem. 33 (1994) 3235–3238. [11] P.J.J.M. Van Ool, H.M. Buck, Recl. Traw. Chim. Pays Bas 100 (1981) 79. [12] J.H. Yu, A.M. Arif, W.G. Bentrude, J. Am. Chem. Soc. 112 (1990) 7451. [13] J.H. Yu, A.E. Sopchik, A.M. Arif, W.G. Bentrude, J. Org. Chem. 55 (1990) 3444. [14] S. Mehdi, J.A. Coderre, J.A. Gerlt, Tetrahedron 39 (1983) 3483. [15] P.M.J. Bburgers, F. Eckstein, D.H. Hunneman, J. Baraniak, R.W. Kinas, K. Lesia, W.J. Stec, J. Biol. Chem. 254 (1979) 9959. [16] J.A. Coderre, S. Mehdi, J.A. Gerlt, J. Am. Chem. Soc. 103 (1981) 1872. [17] P.M. Cullis, R.L. Jarvest, G. Lowe, B.V.L. Potter, J. Chem. Soc. Chem. Commun. (1981) 245. [18] R.L. Jarvest, G. Lowe, J. Baraniak, W.J. Stec, Biochem. J. 203 (1982) 461. [19] A.D. Becke, Phys. Rev. 38 (1988) 3098. [20] C. Lee, W. Yang, R.G. Parr, Development of the Colle–Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B 37 (1988) 785. [21] B. Miehlich, A. Savin, H. Stoll, et al., Chem. Phys. Lett. 157 (1989) 200. [22] A.D. Becke, Density-functional thermochemistry. III The role of exact exchange, J. Chem. Phys. 98 (1993) 5648. [23] (a) C. Gonzalez, H.B. Schlegel, J. Chem. Phys. 90 (1989) 2154; (b) C. Gonzalez, H.B. Schlegel, J. Phys. Chem. 94 (1990) 5523. [24] M.J. Frisch, G.W. Trucks, H.B. Schlegel, et al. GAUSSIAN-98, Gaussian, Inc., Pittsburgh PA, 1998.