Molecular Dynamics and Quantum Chemical Study of Endonuclease

Chapter 33

Molecular Dynamics and Quantum Chemical Study of Endonuclease V Catalytic Mechanism 1

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M . Krauss , N. Luo , R . Nirmala , and R . Osman 1

Center for Advanced Research Biotechnology, National Institute of Science and Technology, Rockville, MD 20850 Department of Physiology and Biophysics, Mt. Sinai School of Medicine, New York, NY 10029

Downloaded by UNIV MASSACHUSETTS AMHERST on August 7, 2012 | http://pubs.acs.org Publication Date: April 8, 1999 | doi: 10.1021/bk-1999-0721.ch033

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Endonuclease V initiates repair of damaged DNA, that contains the thymine dimer, by cleavage of the glycosidic bond through the attack of an amine nucleophile. The transition state for this process is described using a series of model calculations that focus on the electronic characteristics that assist in stabilization of the transition state in the enzyme active site. The inherent geometrical and electronic features of the transition state are obtained in an in vacuo calculation which is then compared to situations where H-bond stabilization of the developing charge is included. The model of the endo V active site includes representations of the Glu-23 and Arg-26 residues. The guanidinium side chain of the arginine residue does not transfer a proton to the thymidine carbonyl in the developing anionic base even when optimizations are intitiated with a proton equidistant between the N of arginine and the O of the base. In the transition state structure, charge separation in the glycosidic bond does not significantly delocalize into the base or sugar, so the stabilization energy due to H-bonding is not large. The activation energy of the glycosidic cleavage catalyzed by a neutral amine is calculated to be about 30 kcal/mol.

1. Introduction Environmental factors, such as high energy radiation, alkylating agents, and UV light, produce a spectrum of damaged DNA which may have severe biological consequences. DNA repair is therefore an essential component for the survival of a biological system. An important category of DNA repair is the multistep process that consists first of base excision followed by the disruption of the strand, whether by aβ-eliminationor the hydrolysis of the phosphodiester bond. The glycosylases that function in this category can be classified into monofunctional and multifunctional enzymes (1,2). Monofunctional glycosylases remove the damaged base and leave an abasic site (AP) (3). The AP site is then processed by another class of enzymes, the AP-endonucleases. In the multifunctional class of enzymes, the bacteriophage T4 endonuclease V (endo V) removes pyrimidine (thymine)

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© 1999 American Chemical Society

In Transition State Modeling for Catalysis; Truhlar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


425 dimers in a combined glycosylase/AP lyase activity. An imino enzyme-DNA intermediate resultsfromthe glycosylase step. This covalent attachment facilitates the catalytic p-elimination of the phosphodiester bond at the abasic site (4,5). Two different nucleophiles have been observed attacking the CI' atom in the sugar, a hydroxyl anion and an amine (2). An example of hydroxyl anion attack is the monofunctional glycosylase, uracil-DNA glycosylase (UDG), which removes uracil from DNA (6-9). Water in the UDG active site may be activated by an Asp yielding the hydroxyl anion nucleophile. An example for the amine, is the Afunctional glycosylase/apyrimidinic (AP) lyase, T4 endonuclease V (endoV), which catalyzes the cleavage of the N-glycosyl bond and the disruption of the phosphodiester bond at the resultant apyrimidinic site. The availability of the crystal structures of the UDG (6-9) and endo V (10,11) enzymes in relevant DNA complexes presents an opportunity to address the question of kinetic selectivity on a fundamental molecular level. As in other enzymes, kinetic selectivity of DNA repair enzymes, as distinguished from a static selectivity of damage recognition, depends on the ability of the enzyme to lower the transition state for the rate determining processes. There have been a number of suggestions that, while the two enzymes differ in the choice of nucleophile, both enhance the hydrolysis of the glycosidic bond by an activating ionic hydrogen bond to the pyrimidine which either prepares the nucleotide for a nucleophilic attack on the sugar or stabilizes the resulting separation of charge during the reaction (2). Activating proton interactions can fundamentally alter or strongly polarize electronic structure and provide an element to the microscopic mechanism that an enzyme is uniquely constructed to deliver. However, while in UDG the reaction terminates at that point, in endoV the apyrimidinic site remains attached to the enzyme to facilitate the lyase step. From kinetic isotope effects, transition state structures have been deduced for hydrolysis of the glycosidic bond in nucleosides (12-14). The transition state is deduced to proceed with little participation of the nucleophile but these studies have considered purine nucleosides that may be initially protonated. This again raises the question of strong hydrogen bonding to the pyrimidine base in endoV or even a protonation of the base. Protonation of the thymidine carbonyl has been suggested for endo V (15). Breaking the glycosidic bond leads to an oxocarbonium cation electronic structure in the sugar and ultimately to the suggestion that substitution of the sugar by a pyrrolidine residue would act as a transition state analogue (16). The electronic character of the transition state in the glycosylase enzymes is required to determine the validity of these analogues. Mutant studies in endoV have established that the terminal amine and a Glu-23 are essential for catalysis but their precise roles in the two steps of the mechanism is not determined (5,15,17-19). The microscopic mechanism has not been determined either for other examples of multifunctional enzymes such as endonuclease III and formamidopyrimidine glycosylase (20). It is interesting to note, however, that prior protonation of a guanine is suggested to activate the glycosidic bond. This note will focus on the glycosylase reaction in endonuclease V but will also examine the inherent characterisitcs of the transition state for the cleavage of the glycosidic bond by the two nucleophiles. The details of the microscopic mechanism for both classes are still not clear regarding the reaction path such as the characteristics


426 of the transition state, the importance of activation of the glycosidic bond prior to or concurrent with nucleophilic attack, and the role played by the Glu-23 in both the glycosylase and lyase steps in endo V. The ab initio quantum chemical determination of the transition states for the in vacuo reactions is straightforward. The in vacuo behavior of the reaction path is very different between the attack of the hydroxyl anion and the amine. It is well known that in the S 2 reaction initiated by an anion in vacuo, the ionic hydrogen or multi-polar bond at long range is sufficiently strong to drive the reaction. In water, the solvation energy of the small hydroxyl anion is greater than that of the transition state that develops leading usually to a substantial activation energy. For the amine attack, there is no ionic interaction and the in vacuo activation energy is already very substantial because of charge separation along the reaction coordinate. Thus, a preliminary analysis of the amine reaction does not require the careful attention to the solvation of the nucleophile even to obtain qualitatively relevant results. At this time the inherent differences between the developing transition state for the hydroxyl and amine attacks will be noted but the hydroxyl reaction will not be followed into the enzyme environment. However, this will be done for the glycosidic cleavage reaction in endonuclease V. Models will be used to analyze the stabilizing influence of the cationic hydrogen bonds to the pyrimidine base but a minimal model of the reaction in endonuclease V will be presented at this time. A molecular dynamics simulation startingfromthe crystal structure of a mutant enzyme-substrate complex is used to reconstruct the wild type enzyme and define a quantum motif for a native enzyme model. Quantum chemical analysis of the reactive behavior at the active site will be investigated by incorporating effective fragment potentials (EFP) to represent those protein residues that are not directly involved in the chemistry but affect the reaction path through their electrostatic interactions or hydrogen bonding. Subsequent to cleavage of the glycosidic bond, a number of proton transfers is required for base product release as well as opening of the sugar ring. The nature of the residues involved in these steps is not clarifed by the crystal structures. Although this preliminary calculation will stop at the cleavage of the glycosidic bond, the final transition state structure should be relevant to subsequent behavior. The transition state structures will be compared to the experimental transition states that have been deducedfromisotopic variations of the rates for various nucleosides. Reaction Path Calculation: The Glycosylase Step Quantum Motif In the multifunctional enzymes the role of the nucleophile is served by an amine, which is an integral part of the protein. In endoV the N-terminal amine plays the role of the nucleophile that attacks the CI' and forms and imino intermediate. The protonation state of the terminal amine is unclear. A self-consistent pK^ calculation (21) of endo V and the endo V-DNA complex determines that the pK^of the amine terminus changes from 8.4 to 7.2 upon DNA binding. Thus, in the complex the nucleophile is about 50% neutral. In the present study we have assumed that the attacking amine terminus is neutral. In the enzyme-DNA structure derivedfromthe MD simulation, Arg-26 is H-bonded to the 0 carbonyl of thymine and Glu-23 is positioned in close proximity to the N-terminus. Mutations have shown Glu-23 to be essential for glycosylase activity . One interpretation emphasizes the stabilization of


N

2



427 the positive charge of the imino intermediate while the other suggests that the negative charge of Glu-23 enhances the nucleophilicity of the amino terminus through a proton transfer mechanism. The importance of stabilization is difficult to reconcile with recent binding experiments demonstrating a positively charged pyrrolidine-based inhibitor does not discriminate between the wild type enzyme and the E23Q mutant (15). Whether the proton transfer mechanism extends to the protonation of the base and activation of the glycosidic bond is also an important consideration in the quantum calculation of the active site reaction path. Only double stranded DNA is a substrate for endo V. The adenine complementary to the 5'-thymine of the dimer flips out of the stacking arrangement and is inserted into a pocket inside the protein. Arg-26 makes a specific hydrogen bond to the 02 position of the 5' thymine. This is unlikely by itself to be the protonating residue that activates the glycosidic bond, analogous to the His-286 in UDG (6), because the calculated pK of this arginine is 11.5. In addition, in vacuo ion-pairs involving protonated arginine have been observed suggesting the difficulty in transferring the proton (22). Although initial protonation has been suggested in the glycosylase step (15), the crystal structure does not obviously reveal the donating residue. Protonation could be concurrent from the terminal amine nucleophile but this amine is found to be on the opposite side of the 5'-thymine with respect to the sugar. In order to obtain the structure of the enzyme environment around the thymine dimer, the enzyme mutant was restored to its native form by replacing Gln-23 with Glu-23. The relaxation of the structure of the complex with classical mechanics after the native protein is restored to Glu-23 is an imperative first step. The molecular dynamics (MD) simulation startsfromthe x-ray structure but transforms Gln-23 back to Glu-23. The complex is embedded in a periodic box of water with a total of 22650 atoms in the system. To equilibrate the water and ions, the system was heated to 600K while keeping the protein-DNA complex in afrozenconformation. A 200ps MD simulation was run at 600K, the system was minimized, reheated to 300K and the constraints on the solute were gradually relaxed over a lOOps time interval. At the end of this process a 700ps trajectory was run on the entire system. AMBER 4.1 was used for all MD calculations (23). The development of the active site H-bonding was examined both through snapshots along the trajectory and a statistical proximity analysis of a given donor or acceptor. For the purpose of constructing the minimal active site model, we note that Oe2 of Glu-23 interacts with the 04' of the sugar through a water about half the time. The water exchanges rapidly within the simulation time. Only one water is used in the active site but a network of waters may be involved. Oe2 also comes close to the nearby terminal amine. Oel of Glu-23 interacts with a water, that does not exchange over the simulation time, and with the backbone amide of Arg-3. The minimal qauntum motif constructedfromthese simulations include, thymidine with a reduced C5-C6 bond to model the dimer and surrounded by abbreviated models for Arg-26, Glu-23, and the Thr-2 terminal amine. Three waters have been included in the model: two H-bonding to Arg-26 and one to Glu-23. This structure is depicted in fig.la. The phosphates were found to be shielded by arginine and waters and will a



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1. Conformations of endonuclease V active site model for: a) reactant, b) 'transition state', c) product. In Transition State Modeling for Catalysis; Truhlar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


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430 be neglected at this time since they do not play an essential role in the cleavage of the glycosidic bond. Quantum Chemistry Methods Optimization of the reactants and the cleavage transition state are all obtained at the RHF level using the GAMESS code (24) with effective core potentials and their concomitant double-zeta level orbitals (25). Since we are interested here in the glycosidic cleavage reaction, only the reactants and transition states are reported here. In vacuo transition states are obtained for both the attack of the hydroxyl anion, ammonia, and methyl amine. Exploration of the effect of hydrogen bonding on the base uses effectivefragmentpotential (EFP) models of protonated and neutral methyl amine bound to one or both carbonyl oxygens on the pyrimidine base as seen in fig. 2. The EFP method is a critical feature of the theoretical model. It is based on the separation of the of the chemical system into two components, a quantum region (QR) and an EFP spectator region (SR). The total Hamiltonian for such a system is defined as the sum of the QR and SR hamiltonians plus an interaction term, V . , H' = H Q + H + V . QR

R

S R

QR

SR

SR

(1) The QR is treated using traditional ab initio methods while the SR is replaced by effective potentials which simulate the quantum interaction between the QR and SR regions. The EFP allows a realistic treatment of the enzymatic reaction in the catalytic active site by representing the protein interactions in the quantum hamiltonian in a computationally tractable method (26,27). The EFP are implemented in the latest versions of the GAMESS code. The EFP accurately represent the electrostatic, polarization, exchange repulsion, and charge transfer effects for non-bonded interactions in the quantum hamiltonian. At the present time the all-electron chemically reacting region can be optimized in the field of thefrozenand constraining EFP or protein environment. Gradient optimization of the quantum region within a fixed EFP environment is implemented in the code. The EFP environment represents the static effective dielectric in a veryfine-grainedmanner. The non-bonded EFP have been shown to be accurate relative to all-electron calculations for the determination of the rotational barrier for an amide (28) and for the optimization of the excited states of formamide solvated by water (29). When the substrate binds the solvent is partially excluded and the reacting region is isolatedfromthe solvent and essentially interior. The MD simulation shows that both the Glu-23 and Arg-26 residues are partially exposed in the enzyme (67% and 48%, respectively) and buried after the substrate binds (100% and 97%, respectively). The electrostatic and polarization components of the EFP are generated by the GAMESS code producing distributed moments through the octupole for the electrostatic EFP (30) and through the dipole for the polarization EFP (31). The points are located at atom and bond mid-points and have been found to accurately represent the fields at hydrogen-bonded distances. The exchange repulsion (ER) and charge transfer (CT) terms are distinct interactions and can be well estimated by the restricted variational space (RVS) methodology (32). The exchange repulsion and charge transfer have similar dependence on overlap between the interacting moieties which allow them to be modeled by the same function. An RVS analysis is used to



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2. Model of H-bonding to 5-methyl, 6-hydro thymidine carbonyls using lysine EFP representation.



432 determine these terms which can be compared well to the difference between the total interaction energy and the sum of the electrostatic and polarizability terms calculated with the EFP. Utility programs are used to fit the exchange repulsion and charge transfer as well as the charge penetration correction to the electrostatic interaction. The repulsive potentials for the pair interactions likely to arise in protein calculations have been generated and are used in the EFP for the Lys, Arg, and Glu. Analysis of Nucleophilic Transition States Nucleophilic attack of a hydoxyl anion was considered in two cases, the neutral and protonated nucleoside. The phosphate was deleted since its charge would prevent the reaction without the protein shielding. The 3' phosphate was first clipped at the oxygen bound to the sugar leaving a 3'OH. The protonated nucleoside behaves differently from the neutral and this is evident in the electronic structure of the reactant molecules. Gradient optimization of the neutral and the protonated uracil nucleoside shows that upon protonation, the glycosidic bond length is increased by 0.04A and the bond order decreases by 20%. Nucleophilic attack of OH" on the CF in the cation nucleoside occurs without a barrier yielding two neutral species with the base in a higher energy tautomer where the proton is located on 02.. For in vacuo nucleophilic attack of an hydroxyl anion on the neutral nucleoside, the sugar 3'OH was reduced to a hydrogen for simplicity. As seen in fig.3a, at the TS the system separates into three charged components with the hydroxyl oxygen, O , 2.26A from CF and in turn CF is separatedfromNI on uracil by 2.02A with an angle of 168 from O-Cl'-N. This is an S 2 transition state which is informative on the electronic character of the sugar and the base as well as their mutual geometric arrangement. The mode behavior of the principle negative eigenvalue shows the concerted motion of the leaving group and nucleophile with respect to the CF in the nucleoside characteristic of an S 2 reaction. The sugar cation has many of the characteristics that have been found in analyzing isotopic rate data (12-14). The calculated CF-04' bond distance is 1.36A which is shorter than the 1.43A calculated in the nucleoside reactant but larger than the 1.25A calculated for the isolated sugar cation. In the isolated cation, the bond order is 1.5 but only 1.1 in the TS. The CI'-04' distance has been deduced to be 1.30A for a TS in a nucleoside hydrolase (12). Stabilization of the TS with aspartate residues has been suggested by either hydrogen-bonding to the ribosyl hydroxyls or interaction with the oxocarbonium cation. However, the atomic charge populations deducedfromthe charge density find that although the ether oxygen is less negative in the TS, it is still appreciably negatively charged with a Mulliken population of -0.38. Any stabilization by an asparatate residue would have to be through a water or metal cation intermediate. The cationic charge is localized on the CI' which is coupled to the localized negative charge of NI on the uracil. The bond orders reflect the geometries. A small bond order of 0.32 is found for the bond intiated between O and CF while the cleaved glycosidic bond is reduced to 0.09. The bond order for CF-04' is only slightly increased to 1.02fromthe 0.94 in the isolated nucleoside. The charge localization on NI is reflected in the bond orders of 1.87 and 1.86, respectively, for the C20 and C40 carbonyl bonds of uracil. There is little charge transferred to the carbonyl bonds and the bond orders of N1-C2 and N1-C6 alter only slightlyfromthe isolated nucleoside. h

N

N



433 Using ammonia as the nucleophile in a simple in vacuo model of the endoV glycosylase step determines a transition state initially with the ribose characterized as an oxocarbonium cation with the Cl'-04' distance of 1.31 A. The separation and geometry between nucleophile, sugar, and base are somewhat different from the hydroxyl anion with the ammonia adopting a tetrahedral conformation consonant with its developing cationic character. This leads to a smaller angle of 134 for N-Cl'-N. The N-Cl' distance is 2.23A similar to that in the anion but the Cl'-N distance to the base of 2.77A is longer. However, a second negative eigenvalue of the Hessian persists with the uracil anion slowly rotating so the carbonyl oxygen can form a long-range electrostatic interaction with the hydrogen on the developing 'ammonium cation'. This interaction is reduced with a methyl amine nucleophile as seen infig.3bbut the overall geometry of the transition state does not alter much. Another very small negative eigenvalue persists because of the weak interaction between the hydrogens on the methyl group with the carbonyl. Both the N-Cl' and Cl'-N distances increase to 2.32A and 2.79A, respectively. Even adding two water EFP to screen the 02 does not sufficiently shield this interaction even though the methyl group is kept about 0.5A farther away and the N-Cl' and Cl'-N distances decrease slightly. The TS geometries and bond orders are summarized in Tables 1 and 2, respectively, for the amine nucleophile. This weak interaction results in a large activation energy of 46 and 39 kcal/mol, respectively for ammonia and methyl amine. Even the slight polarization in methyl amine has an appreciable effect in this calculation. However, these large enthalpic activation energies very much exceed the probable activation energy of about 20 kcal/mol estimatedfromthe observed rate (33). The large activation energy reflects the small interaction between the neutral amine nucleophile and the sugar. While the bond order for the glycosidic bond is decreased below 0.05 signifying almost complete breakage, the N-Cl' bond order is 0.19 in vacuo and 0.28 when the base is H-bonded by a protonated lysine EFP on C20 and a neutral lysine EFP on C40. Stabilization of the charge developing on the base is often noted but the localization of the charge at NI warns us that the ionicity of the carbonyl bonds may not change sufficiently to alter the activation energy appreciably. A cationic arginine residue H-bonds to the C20 of the thymine in the active site of endoV. In the next step of modelling the possibility of H-bond stabilization of the cleavage with the ammonia nucleophile, a cationic H-bond interacts with the C20 carbonyl of uracil using a protonated lysine EFP as described infig.2. As can be seen in Table 1 the changefromthe in vacuo transition state is modest. The nucleophile approaches CI' a little closer and the C20 bond has stretched due to the interaction with the ionic H-bond. Although the interaction has not substantially altered the localization of the charge at NI in the base, it does yield a modest reduction in the activation energy of about 8 kcal/mol. This is due to the non-linear H-bond that resulted after optimization. The proton donates into the maximum of the oxygen electron density which would be in a ring of about 30° awayfromlinear. Nonetheless, the activation energy is still very high. Adding a neutral lysine EFP to C40 also yields a reduction in the activation energy of 8 kcal/mol. The neutral lysine interaction does not contribute as the distance between N-C4 increases to 5.49A during optimization while the N-C2 value goes to 2.72A compared to 2.80A when there is



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3. Conformations of in vacuo transition states for the nucleophiles: a) hydroxyl anion, b) methyl amine.


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Table 1. Transition State Geometries.

1. 2. 3. 4. 5.

N-Cl'

Cl'-Nl

2.234 2.317 2.016 2.085 2.935

2.769 2.792 2.689 2.652 2.551

Cr-04 N1-C2 N1-C6 C2-0 C4-0 H-C20H-C40 1.308 1.306 1.326 1.319 1.290

1.360 1.360 1.338 1.342 1.315

1.374 1.373 1.390 1.386 1.468

1.279 1.278 1.295 1.293 1.290

1.249 1.248 1.240 1.243 1.242

1.811 1.966 1.978

1. in vacuo ammonia, all geometries in A. 2. in vacuo methyl amine 3. lysp EFP interacting with C20 4. lysp, lys EFP interacting with C20 and C40, respectively 5. model of endo V active space with arg and glu efp Table 2. Bond Orders in Bonds Formed or Altered by Glycosidic Cleavage N-Cl' Cl'-Nl 1. 0.68 2. 0.19 3. 0.13 4. 0.28 5.

Molecular Dynamics and Quantum Chemical Study of Endonuclease

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