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DFT study of the reaction proceeding in the cytidine deaminase
Chemical Physics Letters 381 (2003) 660–665 www.elsevier.com/locate/cplett
DFT study of the reaction proceeding in the cytidine deaminase Pawel Kedzierski a, W. Andrzej Sokalski a,*, Hansong Cheng b, John Mitchell b, Jerzy Leszczynski c a
Molecular Modeling Laboratory, Institute of Physical and Theoretical Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland b Air Products and Chemicals Inc., Allentown, PA 18195-1501, USA c Computational Center for Molecular Structure and Interactions, Department of Chemistry, Jackson State University, MS 39217, USA Received 11 August 2003; in final form 30 September 2003 Published online: 4 November 2003
Abstract The deamination of cytidine performed by cytidine deaminase has been modeled using density functional theory yielding a reaction mechanism differing from that previously reported using semiempirical results. The main difference consists in the initial reaction step starting with Zn-bound water and N3-protonated cytidine. This study allows for the first time to close complete reaction cycle for the investigated process. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction Cytidine and deoxycitidine deaminases (CDA, EC 3.5.4.5, 3.5.4.14) are rather conservative 54 kDa homodimer enzymes utilizing one Zn2þ cation per monomer for catalysis. They are considerably efficient, accelerating the rate of hydrolytic deamination of cytidine by about 11 orders of magnitude [1,2]. These enzymes are important in nucleotide metabolism and are both medically and industrially
*
Corresponding author. Fax: +48-71-3203364. E-mail address: [email protected] (W.A. Sokalski).
interesting. CDA is responsible for degradation of cytidine-based antitumour agents [3] as it converts relatively nontoxic 5-fluorocytosine into 5-fluorouracil used in enzyme-prodrug gene therapy [4] and is a plausible catalyst for the enantioselective deamination [5]. Another class of cytidine deaminases induces G!A hypermutation in newly synthesised viral DNA and is the main driving force for the generation of drug resistance [6,7]. The details of catalytic function of CDA may yield essential clues for conversion of alcohols and olefins to amines as well as in assisting asymmetric synthesis providing important alternative to conventional chemical technologies. The experimental efforts, aimed at
0009-2614/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2003.10.042
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
explanation of the CDA catalysis, date back to the seventies [8–10]. The role of the active site residues seems to be well recognized, and the reaction energetics are supported by evidence from accurate kinetics and site-directed mutagenesis experiments [11,12]. Several structures of CDAs are available from the Protein Data Bank [13–18]. The above described pool of the published data provides the necessary initial information for theoretical studies. Nevertheless, the microscopic mechanism of the catalytic action with this enzyme is still not fully explained. The two most up-to date computational studies of the CDA active site by Lewis et al. [19,20] provide specific structures of the substrates. However, some alternative protonation states of the reaction model, which seem plausible, were not evaluated by Lewis et al., and the stationary points obtained using the semiempirical approach may not always be conclusive [21,22]. The aim of this work was to provide more complete description of the reaction mechanism using density functional theory. In the course of this study, in order to explain experimental findings it was found necessary to sample protonation states of the system other than those proposed so far [19], exploring alternative reaction pathways.
661
2. Methods The initial structures of the reaction models were based upon the crystal structure of CDAinhibitor complex 1CTT from the Protein Data Bank [13]. The residues which were represented in our reaction models are His 102, Cys 129 and Cys 132, which coordinate the catalytic Zn2þ cation, the zinc-bound water molecule, and the general acid–base Glu 104. Due to solvent exclusion from the active site of CDA [13], a limited, gas-phase active site model was considered appropriate for this investigation. Searches for transition states were performed from various starting points using eigenvalue-following, QST2 and QST3 methods implemented in Gaussian software [23]. All the stationary structures were optimized using Perdew–Wang 91 density functional and Lanl2DZ basis set for all atoms. The structures were verified by harmonic vibrational frequency analysis. The energy differences reported include zero point energy (ZPE) and thermal corrections at 298 K. Some results were obtained using PM3 semiempirical method, for comparison with the published data [19]. They are described in the following section.
Fig. 1. Conversion of methylamine to methanol on the model of cytidine deaminase prosthetic group: (a) the model and (b) reaction path.
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P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
The Molden package [24] was employed for model preparation and for visualization of the results.
3. Results and discussion Investigation of the deamination pathway started with a limited model presented in Fig. 1. The substrate complex and the transition state calculated with this model are separated by the energy of 202.8 kJ/mol. However, half of this energy difference is due to the reorientation of the reagents and the formation of hydrogen bonds. Analogous to the model proposed by Lewis et al. [19], the ground state of the system should involve the water molecule dissociated into Zn2þ -bound OH and the proton from the active site acid (Glu 104). A similar structure was observed in our study halfway along the reaction path (Fig. 1b), but it was not a local minimum. In the work by Lewis et al. [19], a large active site model was employed, which provided both an adequate description of the reaction environment and the spatial constraints imposed by the enzyme. However, it was evaluated using PM3 level of theory. The discrepancy observed between PM3 results and present study might be due to the limited model applied, or it can be an artifact of the semiempirical approximation. In order to test it we investigated the behaviour of model system including a representation of the enzyme prostethic group, Zn2þ (SH )2 (NH3 ), a catalytic water molecule, and formate, representing the active site residue Glu 104 (Fig. 2). A relaxed scan of the potential energy surface along the O H distance was performed using PM3 and Perdew–Wang density functional methods. For verification of the lowest energy pathway, the scan was performed in two directions: 1. Shifting the proton away from the water molecule oxygen, and 2. Starting with the proton on the formic acid side and pulling it away from the carboxyl oxygen. The potential surface scans in both directions revealed consistent results. With the density functional approach one stable configuration of the system is observed (Fig. 2a), whereas PM3 method
Fig. 2. Behaviour of the activated water–acid proton transfer using PW91 density functional (a) and PM3 (b).
reveals two local minima (Fig. 2b). The optimal configuration for both methods appears to be the one with undissociated water, and the existence of additional dissociated states seems to be an artifact of the PM3 approximation. Another explanation of the obtained reaction barrier shape was also considered. Starting the reaction from the Zn2þ (OH ) species as proposed in [20] implies the nucleophylic attack of the hydroxyl oxygen on the C4 atom of uridine. This should be inhibited by the p-electron distribution, but facilitated by the positive charge on the cytosine ring, which suggests that the cytidine should possibly be protonated. This idea is consistent with the optimum of CDA activity being at low pH values. There are two possible protonation sites of cytosine, at the –NH2 group and at the N3 atom. We
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
found, however, that the additional proton from –NHþ 3 slips towards the zinc-bound OH– and restores the water molecule. The other protonation site at N4 seems to be more likely, but protonation at N4 implied reversal of the hydrogen-bond to Glu 104 and therefore from Glu 104 to Zn-bound water. The ground state for the CDA active site model was therefore assumed to involve undissociated, Zn2þ bound water. With this provision a larger reaction model was constructed (Fig. 3). This
663
model represents active site cysteines 129 and 132 as sulfhydryl groups and histidine 102 as methylimine. These groups coordinate the catalytic Zn2þ cation. The fourth coordination position is occupied by the water molecule. The cytidine substrate was represented by cytosine ring (Fig. 3). This model was utilized to search for possible transition states. The lowest energy transition state found is presented in Fig. 4. It features the attack of the zinc-activated water oxygen to the C4 carbon
Fig. 3. Proposed reaction pathway for cytidine deaminase. (a) Binding of water to the zinc center, (b) protonation of the cytosine ring on N3 by active-site acid, (c) attack of the zinc-activated water molecule on cytosine C4–NH2 and (d) deprotonation and decay of the C4 geminal phenylamine intermediate to uracil and ammonia.
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P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
of cytosine, coupled with proton transfer from the water to the C4 amino group. No separate transition state was found for the HOH H2 N-proton transfer, nor was there a local minimum for the HO þ H3 N-configuration. The formation of the transition state is favoured by protonation of cytosine at the N3 nitrogen. The obvious candidate providing the proton for N3 is the active site glutamate 104, whose pKa is similar to pKa of cytosine at N3. This proton transfer should occur readily and it is not expected to be the reaction bottleneck. The transition state presented in Fig. 4a decays to an intermediate product with geminal OH and NHþ 3 substituents at C4 (Fig. 4b). This structure occupies a shallow local minimum and it decomposes readily into uracil and ammonia upon deprotonation of the C4–OH group. The energy
H HN
difference between the intermediate and the product is 66.5 kJ/mol with ZPE and thermal corrections. In order to simulate the presence of the active site base Glu 104 , the model was extended with a carboxyl group. The –COO group was attached via a spacer –CH2 – to one of the sulfhydryl groups (Fig. 5a). Such arrangement reduces the number of degrees of freedom of the system while keeping the acid at a distance similar to Glu 104 N3 distance within the CDA active site. Using this model, we observed barrierless deprotonation of the C4 geminal intermediate and subsequent barrierless decay yielding uracil and ammonia (Fig. 5b). The most comprehensive theoretical studies of the CDA active site were published by Lewis et al. [19,20]. They studied a large active site model, but structure optimization had been done using PM3
H
H
H
O
NH
O H C
C
H H
H
C
H
N
H H
S
C
S
H
C N
H
S
H
(a)
H
C
Zn
S
N O
H
H
N
C
H
O
Zn
H
C
N
H
N H
C
(b)
H
H
C H
Fig. 4. The transition state (a) and the C4 geminal phenylamine intermediate of the cytosine deamination rate-limiting step. The energy difference between transition state (a) and the product (b) is )0.025274 hartree ()66.5 kJ/mole) including ZPE and thermal corrections (at 298 K).
H H N H
H
O
O
O
H C H H C N
C
H
H
C N
H
Zn
S
C N H
C O
N
H
H
C
(b)
H
S
C
S
N
H
(a)
H
H
H Zn
C
C C
H
H
H
O H
H
O
C O
H O
C S
H N
HN
C
H
H
Fig. 5. Spontaneous deamination of the C4-aminouracil intermediate (a) upon presence of a base. The –CH2 –COO group was anchored to one of the sulphur atoms in order to reduce the number of degrees of freedom in the system. During optimization, the proton is transferred to the –COO group and the ammonia molecule separates from the uracil ring (b).
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
Hamiltonian. In addition, the evaluation of possible protonation states was not exhaustive. When we employed the substrate structure proposed in [19] in our study, we found that it leads to an unacceptably high reaction barrier. Considering the discrepancy between the PM3 and DFT results in this case and the behaviour of differently protonated models, we proposed an alternative ground state configuration of the system. This configuration featuring Zn-bound water and N3protonated cytidine allows us to propose a consistent and complete reaction pathway for CDA (Fig. 3). Starting with an empty active site, the reaction begins with binding a water molecule at the zinc cation, and cytidine (Fig. 3a). Upon binding of the cytidine substrate, protonation state of the cytosine ring at N3 should reach a rapid equilibrium with respect to the proton exchange with glutamate 104, since both have almost equal acidity (Fig. 3b). Protonated form of cytidine reacts with the zinc-activated water molecule forming an unstable geminal intermediate at C4. In the presence of the Glu 104 base, this intermediate decays to products, restoring the initial protonation state of this residue. Dissociation of the products completes the reaction cycle. Structural data used in this work are available from the authors upon request.
4. Conclusions The reaction catalysed by CDA was investigated using density functional theory. For the first time, the complete reaction cycle is proposed. The early stage of the reaction mechanism was found to be different from that proposed earlier [19,20] using a semiempirical approach. The relevant stationary points on the reaction pathway were characterized.
Acknowledgements We thank Dr. Robert Higgins for invaluable comments on the reaction mechanism and for proof-reading of the manuscript. This work has
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been partially supported by Air Products and Chemicals and Wroclaw University of Technology as well as ONR grant no. b00014-98-1-0592, NSFCREST grant no. 9805465 and 9706268, and NSFEPSCoR grant no. 300423-190200-21000. References [1] H. Hosono, S. Kuno, J. Biochem. 74 (1973) 797. [2] L. Frick, J.P. MacNeela, R. Wolfenden, Bioorg.Chem. 15 (1987) 100. [3] B. Chandrasekaren, R.L. Capizzi, T.E. Kute, T. Morgan, J. Dimling, Cancer Res. 49 (1989) 3259. [4] O. Greco, G.U. Dachs, J. Cell. Physiol. 187 (2001) 22. [5] J.H. Woo, H.J. Shin, T.H. Kim, S.Y. Ghim, L.S. Jeong, J.G. Kim, B.H. Song, Biotech. Lett. 23 (2) (2001) 131. [6] S.K. Petersen-Mahrt, R.S. Harris, M.S. Neuberger, Nature 418 (2002) 99. [7] H. Zhang, B. Yang, P.J. Pomerantz, C.M. Zhang, S.C. Arunachalam, L. Gao, Nature 424 (2003) 94. [8] R.M. Cohen, R. Wolfenden, J. Biol. Chem. 246 (1971) 7561. [9] D.F. Wentworth, R. Wolfenden, Biochemistry 14 (1975) 5099. [10] B.E. Evans, G.N. Mitchell, R. Wolfenden, Biochemistry 14 (1975) 621. [11] D.C. Carlow, A.A. Smith, C.C. Yang, S.A. Short, R. Wolfenden, Biochemistry 34 (1995) 4220. [12] D.C. Carlow, R. Wolfenden, Biochemistry 37 (34) (1998) 11873. [13] S. Xiang, S.A. Short, R. Wolfenden, C.W. Carter Jr., Biochemistry 34 (1995) 4516. [14] S. Xiang, S.A. Short, R. Wolfenden, C.W. Carter Jr., Biochemistry 35 (1996) 1335. [15] S. Xiang, S.A. Short, R. Wolfenden, C.W. Carter Jr., Biochemistry 36 (1997) 2563. [16] E. Johansson, N. Mejlhede, J. Neuhard, S. Larsen, Biochemistry 41 (2002). [17] G.C. Ireton, G. McDermott, M.E. Black, B.L. Stoddart, J. Mol. Biol. 315 (2002) 687. [18] T.P. Ko, J.J. Lin, Y. Hu, Y.H. Hsu, A.H.J. Wang, S.H. Liaw, J. Biol. Chem. 278 (2003) 19111. [19] J.P. Lewis, C.W. Carter Jr., J. Hermans, W. Pan, T.S. Lee, W. Yang, J. Am. Chem. Soc. 120 (22) (1998) 5407. [20] J.P. Lewis, S. Liu, T.S. Lee, W. Yang, J. Comput. Phys. 151 (1999) 242. [21] B.S. Jursic, Z. Zdravkovski, J. Heterocycl. Chem. 31 (1994) 1429. [22] P.L. Cummins, S.P. Greatbanks, A.P. Rendell, J.E. Gready, J. Phys. Chem. B 106 (2002) 9934. [23] J.A. Pople et al., Gaussian 98, Revision A.11.3, Gaussian, Inc.,, Pittsburgh PA, 2002. [24] G. Schaftenaar, J.H. Noordik, J. Comput. Aided Mol. Design 14 (2000) 123.
DFT study of the reaction proceeding in the cytidine deaminase Pawel Kedzierski a, W. Andrzej Sokalski a,*, Hansong Cheng b, John Mitchell b, Jerzy Leszczynski c a
Molecular Modeling Laboratory, Institute of Physical and Theoretical Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland b Air Products and Chemicals Inc., Allentown, PA 18195-1501, USA c Computational Center for Molecular Structure and Interactions, Department of Chemistry, Jackson State University, MS 39217, USA Received 11 August 2003; in final form 30 September 2003 Published online: 4 November 2003
Abstract The deamination of cytidine performed by cytidine deaminase has been modeled using density functional theory yielding a reaction mechanism differing from that previously reported using semiempirical results. The main difference consists in the initial reaction step starting with Zn-bound water and N3-protonated cytidine. This study allows for the first time to close complete reaction cycle for the investigated process. Ó 2003 Elsevier B.V. All rights reserved.
1. Introduction Cytidine and deoxycitidine deaminases (CDA, EC 3.5.4.5, 3.5.4.14) are rather conservative 54 kDa homodimer enzymes utilizing one Zn2þ cation per monomer for catalysis. They are considerably efficient, accelerating the rate of hydrolytic deamination of cytidine by about 11 orders of magnitude [1,2]. These enzymes are important in nucleotide metabolism and are both medically and industrially
*
Corresponding author. Fax: +48-71-3203364. E-mail address: [email protected] (W.A. Sokalski).
interesting. CDA is responsible for degradation of cytidine-based antitumour agents [3] as it converts relatively nontoxic 5-fluorocytosine into 5-fluorouracil used in enzyme-prodrug gene therapy [4] and is a plausible catalyst for the enantioselective deamination [5]. Another class of cytidine deaminases induces G!A hypermutation in newly synthesised viral DNA and is the main driving force for the generation of drug resistance [6,7]. The details of catalytic function of CDA may yield essential clues for conversion of alcohols and olefins to amines as well as in assisting asymmetric synthesis providing important alternative to conventional chemical technologies. The experimental efforts, aimed at
0009-2614/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2003.10.042
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
explanation of the CDA catalysis, date back to the seventies [8–10]. The role of the active site residues seems to be well recognized, and the reaction energetics are supported by evidence from accurate kinetics and site-directed mutagenesis experiments [11,12]. Several structures of CDAs are available from the Protein Data Bank [13–18]. The above described pool of the published data provides the necessary initial information for theoretical studies. Nevertheless, the microscopic mechanism of the catalytic action with this enzyme is still not fully explained. The two most up-to date computational studies of the CDA active site by Lewis et al. [19,20] provide specific structures of the substrates. However, some alternative protonation states of the reaction model, which seem plausible, were not evaluated by Lewis et al., and the stationary points obtained using the semiempirical approach may not always be conclusive [21,22]. The aim of this work was to provide more complete description of the reaction mechanism using density functional theory. In the course of this study, in order to explain experimental findings it was found necessary to sample protonation states of the system other than those proposed so far [19], exploring alternative reaction pathways.
661
2. Methods The initial structures of the reaction models were based upon the crystal structure of CDAinhibitor complex 1CTT from the Protein Data Bank [13]. The residues which were represented in our reaction models are His 102, Cys 129 and Cys 132, which coordinate the catalytic Zn2þ cation, the zinc-bound water molecule, and the general acid–base Glu 104. Due to solvent exclusion from the active site of CDA [13], a limited, gas-phase active site model was considered appropriate for this investigation. Searches for transition states were performed from various starting points using eigenvalue-following, QST2 and QST3 methods implemented in Gaussian software [23]. All the stationary structures were optimized using Perdew–Wang 91 density functional and Lanl2DZ basis set for all atoms. The structures were verified by harmonic vibrational frequency analysis. The energy differences reported include zero point energy (ZPE) and thermal corrections at 298 K. Some results were obtained using PM3 semiempirical method, for comparison with the published data [19]. They are described in the following section.
Fig. 1. Conversion of methylamine to methanol on the model of cytidine deaminase prosthetic group: (a) the model and (b) reaction path.
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P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
The Molden package [24] was employed for model preparation and for visualization of the results.
3. Results and discussion Investigation of the deamination pathway started with a limited model presented in Fig. 1. The substrate complex and the transition state calculated with this model are separated by the energy of 202.8 kJ/mol. However, half of this energy difference is due to the reorientation of the reagents and the formation of hydrogen bonds. Analogous to the model proposed by Lewis et al. [19], the ground state of the system should involve the water molecule dissociated into Zn2þ -bound OH and the proton from the active site acid (Glu 104). A similar structure was observed in our study halfway along the reaction path (Fig. 1b), but it was not a local minimum. In the work by Lewis et al. [19], a large active site model was employed, which provided both an adequate description of the reaction environment and the spatial constraints imposed by the enzyme. However, it was evaluated using PM3 level of theory. The discrepancy observed between PM3 results and present study might be due to the limited model applied, or it can be an artifact of the semiempirical approximation. In order to test it we investigated the behaviour of model system including a representation of the enzyme prostethic group, Zn2þ (SH )2 (NH3 ), a catalytic water molecule, and formate, representing the active site residue Glu 104 (Fig. 2). A relaxed scan of the potential energy surface along the O H distance was performed using PM3 and Perdew–Wang density functional methods. For verification of the lowest energy pathway, the scan was performed in two directions: 1. Shifting the proton away from the water molecule oxygen, and 2. Starting with the proton on the formic acid side and pulling it away from the carboxyl oxygen. The potential surface scans in both directions revealed consistent results. With the density functional approach one stable configuration of the system is observed (Fig. 2a), whereas PM3 method
Fig. 2. Behaviour of the activated water–acid proton transfer using PW91 density functional (a) and PM3 (b).
reveals two local minima (Fig. 2b). The optimal configuration for both methods appears to be the one with undissociated water, and the existence of additional dissociated states seems to be an artifact of the PM3 approximation. Another explanation of the obtained reaction barrier shape was also considered. Starting the reaction from the Zn2þ (OH ) species as proposed in [20] implies the nucleophylic attack of the hydroxyl oxygen on the C4 atom of uridine. This should be inhibited by the p-electron distribution, but facilitated by the positive charge on the cytosine ring, which suggests that the cytidine should possibly be protonated. This idea is consistent with the optimum of CDA activity being at low pH values. There are two possible protonation sites of cytosine, at the –NH2 group and at the N3 atom. We
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
found, however, that the additional proton from –NHþ 3 slips towards the zinc-bound OH– and restores the water molecule. The other protonation site at N4 seems to be more likely, but protonation at N4 implied reversal of the hydrogen-bond to Glu 104 and therefore from Glu 104 to Zn-bound water. The ground state for the CDA active site model was therefore assumed to involve undissociated, Zn2þ bound water. With this provision a larger reaction model was constructed (Fig. 3). This
663
model represents active site cysteines 129 and 132 as sulfhydryl groups and histidine 102 as methylimine. These groups coordinate the catalytic Zn2þ cation. The fourth coordination position is occupied by the water molecule. The cytidine substrate was represented by cytosine ring (Fig. 3). This model was utilized to search for possible transition states. The lowest energy transition state found is presented in Fig. 4. It features the attack of the zinc-activated water oxygen to the C4 carbon
Fig. 3. Proposed reaction pathway for cytidine deaminase. (a) Binding of water to the zinc center, (b) protonation of the cytosine ring on N3 by active-site acid, (c) attack of the zinc-activated water molecule on cytosine C4–NH2 and (d) deprotonation and decay of the C4 geminal phenylamine intermediate to uracil and ammonia.
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P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
of cytosine, coupled with proton transfer from the water to the C4 amino group. No separate transition state was found for the HOH H2 N-proton transfer, nor was there a local minimum for the HO þ H3 N-configuration. The formation of the transition state is favoured by protonation of cytosine at the N3 nitrogen. The obvious candidate providing the proton for N3 is the active site glutamate 104, whose pKa is similar to pKa of cytosine at N3. This proton transfer should occur readily and it is not expected to be the reaction bottleneck. The transition state presented in Fig. 4a decays to an intermediate product with geminal OH and NHþ 3 substituents at C4 (Fig. 4b). This structure occupies a shallow local minimum and it decomposes readily into uracil and ammonia upon deprotonation of the C4–OH group. The energy
H HN
difference between the intermediate and the product is 66.5 kJ/mol with ZPE and thermal corrections. In order to simulate the presence of the active site base Glu 104 , the model was extended with a carboxyl group. The –COO group was attached via a spacer –CH2 – to one of the sulfhydryl groups (Fig. 5a). Such arrangement reduces the number of degrees of freedom of the system while keeping the acid at a distance similar to Glu 104 N3 distance within the CDA active site. Using this model, we observed barrierless deprotonation of the C4 geminal intermediate and subsequent barrierless decay yielding uracil and ammonia (Fig. 5b). The most comprehensive theoretical studies of the CDA active site were published by Lewis et al. [19,20]. They studied a large active site model, but structure optimization had been done using PM3
H
H
H
O
NH
O H C
C
H H
H
C
H
N
H H
S
C
S
H
C N
H
S
H
(a)
H
C
Zn
S
N O
H
H
N
C
H
O
Zn
H
C
N
H
N H
C
(b)
H
H
C H
Fig. 4. The transition state (a) and the C4 geminal phenylamine intermediate of the cytosine deamination rate-limiting step. The energy difference between transition state (a) and the product (b) is )0.025274 hartree ()66.5 kJ/mole) including ZPE and thermal corrections (at 298 K).
H H N H
H
O
O
O
H C H H C N
C
H
H
C N
H
Zn
S
C N H
C O
N
H
H
C
(b)
H
S
C
S
N
H
(a)
H
H
H Zn
C
C C
H
H
H
O H
H
O
C O
H O
C S
H N
HN
C
H
H
Fig. 5. Spontaneous deamination of the C4-aminouracil intermediate (a) upon presence of a base. The –CH2 –COO group was anchored to one of the sulphur atoms in order to reduce the number of degrees of freedom in the system. During optimization, the proton is transferred to the –COO group and the ammonia molecule separates from the uracil ring (b).
P. Kedzierski et al. / Chemical Physics Letters 381 (2003) 660–665
Hamiltonian. In addition, the evaluation of possible protonation states was not exhaustive. When we employed the substrate structure proposed in [19] in our study, we found that it leads to an unacceptably high reaction barrier. Considering the discrepancy between the PM3 and DFT results in this case and the behaviour of differently protonated models, we proposed an alternative ground state configuration of the system. This configuration featuring Zn-bound water and N3protonated cytidine allows us to propose a consistent and complete reaction pathway for CDA (Fig. 3). Starting with an empty active site, the reaction begins with binding a water molecule at the zinc cation, and cytidine (Fig. 3a). Upon binding of the cytidine substrate, protonation state of the cytosine ring at N3 should reach a rapid equilibrium with respect to the proton exchange with glutamate 104, since both have almost equal acidity (Fig. 3b). Protonated form of cytidine reacts with the zinc-activated water molecule forming an unstable geminal intermediate at C4. In the presence of the Glu 104 base, this intermediate decays to products, restoring the initial protonation state of this residue. Dissociation of the products completes the reaction cycle. Structural data used in this work are available from the authors upon request.
4. Conclusions The reaction catalysed by CDA was investigated using density functional theory. For the first time, the complete reaction cycle is proposed. The early stage of the reaction mechanism was found to be different from that proposed earlier [19,20] using a semiempirical approach. The relevant stationary points on the reaction pathway were characterized.
Acknowledgements We thank Dr. Robert Higgins for invaluable comments on the reaction mechanism and for proof-reading of the manuscript. This work has
665
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