Quantum Chemical Investigation of Enzymatic Activity in DNA Polymeraseâ. AMechanistic Study

Yuri G. Abashkin,* John W. Erickson, and Stanley K. BurtAdVanced Biomedical Computing Center and Structural Biochemistry Program, Frederick Cancer Researchand DeVelopment Center, NCI/SAIC Frederick, P.O. Box B, Frederick, Maryland 21702-1201

ReceiVed: October 4, 2000

Recent experimental observations support the assumption that all families of polynucleotide polymeraseshave a universal “two-metal-ion” mechanism of nucleotide addition. This mechanism provides a general pictureof the nucleotidyl transfer reaction. However, the detailed reaction pathway is still a matter of debate. Weinvestigated two potential reaction pathways for DNA polymeraseâ using density-functional theory. Ourmodel consists of 67 atoms of the polymerase active site and includes all major features thought to be importantfor catalysis. The first mechanism we investigated involves the formation of a PO3 intermediate. Thisintermediate is thought to be involved in phosphate reactions in solution and could be accommodated in thepolymeraseâ active site. However, the barrier to formation of this intermediate is 37.0 kcal/mol, and we donot expect that this mechanism is the one that occurs in the enzyme. The second mechanism that leads to apentacoordinated intermediate appears to be feasible. This stepwise mechanism has relatively low barriersand, after the nucleophilic attack, every step of the reaction is exothermic. The rate-limiting step of the reactionis the nucleophilic attack, which needs 13 kcal/mol of activation energy. We predict that the barrier of thecorresponding transition state, which is ionic, can be further lowered by taking into account electrostaticstabilization coming from the rest of the protein.

Introduction

An understanding of the mechanisms of DNA polymerasesis of fundamental and practical importance. Replication ofgenomic DNA is essential for evolution and the maintenanceof life.1 Practical benefits of knowing the DNA mechanismscan be found in the field of drug design. Inhibition of a host-cell polymerase that affects genomic replication can be fatal toan organism, whereas inhibition of HIV-1 reverse transcriptase(RT) prevents viral replication in host cells. A detailedunderstanding of the mechanisms of viral and host-cell poly-merases may lead to the design of less toxic HIV-1 RTinhibitors.

Recently obtained crystal structures of DNA polymerase ofbacteriophage T72 and bacterial DNA polymerase3 stronglysupport the assumption that all families of polynucleotidepolymerases have a universal ‘two-metal-ion’ mechanism ofnucleotide addition.4 Indeed, two magnesium ions, which arebound to two aspartic residues, are widely conserved amongDNA and RNA polymerases.5-8 Thus, findings on a mechanismof some polymerase might be applicable to other polymerasesbecause it appears that the mechanism is conserved in evolution.The two-metal-ion mechanism provides a general picture of thenucleotidyl transfer reaction. However, the detailed reactionpathway is still a matter of debate.4,9,10In this paper, we considerpossible reaction pathways in the framework of the two-metal-ion approach for DNA polymeraseâ (Pol â).9

A schematic representation of the DNA Polâ active site andbasic features of the nucleotidyl transfer reaction are presentedin Scheme 1. In the course of the reaction, a bond between theoxygen atom of the 3′-OH group of the template primer andthe PR atom of an incoming 2′-deoxyribonucleoside 5′-triph-osphate (dNTP) is formed. This leads to the elongation of the

primer by one new base. The cleavage of the PR-OPâ bondand elimination of the pyrophosphate (Pâ-Pγ moiety) are thenneeded to complete the DNA elongation. According to the two-metal-ion mechanism,4 the Mg1 ion lowers the affinity of thehydrogen of the 3′-OH group. This facilitates creation of anattacking nucleophile. The Mg2 ion assists the leaving of thepyrophosphate. Both ions are thought to stabilize the proposedpentacoordinated intermediate that is created after the nucleo-philic attack.

In this paper, we use density-functional theory (DFT) to studythe catalytic mode of action of DNA Polâ. DFT calculationsthat include nonlocal corrections have been shown to be practicalfor describing large biological systems where hydrogen bondingand proton transfer are important.11 The next section describesthe theoretical method used in this investigation and is followedby a description of the active site model. Discussion of theresults and a conclusion section complete the paper.

SCHEME 1

287J. Phys. Chem. B2001,105,287-292

10.1021/jp003629x CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 12/02/2000

Method

All calculations were carried out using the DGauss program.12

The DGauss code implemented in UniChem 3.0 version wasused.13 The calculations were performed using the DZVP basissets which are (621/41/1) for carbon, oxygen, and nitrogenatoms; (41) for hydrogen atoms; and (6321/411/1) for the Mgatom.14,15The nonlocal corrections using the Becke exchange16

and Perdue correlation17 potentials have been obtained startingfrom the Vosko, Wilk, and Nuisar local potential.18 Theconvergence of geometry optimization was less than 0.001hartree/b for the largest Cartesian energy gradient component.For every stationary point, a Hessian matrix was calculated toprove the nature of the optimized structure. The matrix of secondderivatives was evaluated analytically.19

Construction of a Model for the Pol â Active Site.On thebasis of the crystal structure of Kraut and co-workers,9,20 weconstructed a model of the ternary complex of Polâ, a templateprimer, and a substrate (Scheme 1; Figure 1). All residuesthought to be important for the catalytic activity or substratebinding were explicitly or implicitly included.

In our model of the Polâ active site, two divalent magnesiumions (Mg1 and Mg2) are ligated to the enzyme by two formatecarboxyl groups that represent aspartic acid residues: Asp-190and Asp-192. Following the analysis of the crystal structurepresented by Pelletier et al.,9 we placed a water molecule incoordination to the Mg1 atom. In addition, Asp-256 is locatedapproximately 3.0 Å from the Mg1 ion. This large distancebetween the positively charged ion and the negatively chargedAsp-256 implies that the aspartic residue probably interacts withthe metal ion indirectly through the water molecule. We modeledthe aspartate-water group as a hydroxyl anion that is attachedto the Mg1 atom and is oriented toward the experimentallydetermined position of Asp-256.

The second metal ion in the active site, Mg2, was experi-mentally shown to coordinate a water molecule, Asp-190, Asp-192, and several oxygen atoms of the substrate. Our preliminarycalculations showed that the absence of this water molecule fromthe model significantly influenced the geometrical parametersof the two-metal-fragment (Mg1-Asp-190-Asp-191-Mg2)coordination. It was previously shown21 that water moleculesalso play an important role in the thermodynamics of magnesiumbinding to proteins. Therefore, we included this water moleculeto maintain the structural and thermodynamic integrity of themodel.

We modeled the dNTP substrate as a triphosphate compoundwith a methyl group substituting for the deoxyribonucleosidepart of dNTP. In solution, the triphosphate carries at least onenegative charge on every phosphate (R, â, and γ). In theenzymatic environment of the Polâ active site, two of thephosphates (Pâ and Pγ) are neutralized by the positive chargesfrom Arg-183 and Arg-149. These two enzyme residues stronglyinteract with the substrate, resulting in the formation of twoshort hydrogen bonds: the distance between a nitrogen of Arg-183 and an oxygen of theâ phosphate is 2.7 Å, and the distancebetween a nitrogen of Arg-149 and an oxygen of theγ phosphateis 2.5 Å. The polar side chain of Ser-180 is also in the vicinityof the â andγ phosphates. Obviously, the arginine and serineresidues play a very important role in the substrate binding andstabilization (and possibly distribution) of the substrate charges.Our model of the Polâ active site, depicted in Figure 1, includesthe appropriate functional groups that mimic the serine andarginine side chains, a CH3OH compound for Ser-180 and CH3-(NH2)2

+ for Arg-183 and Arg-149. Additional stabilization ofthe substrate negative charges comes from the two magnesiumions: Mg1 is situated 2.7 Å away of an oxygen of theRphosphate; Mg2 is coordinated by oxygens of theâ and γphosphates (1.7 Å and 2.3 Å, correspondingly).

The neutralization of the third charge on the substrate is dueto the sharing of a proton between the PR oxygen (O1) and anoxygen of Asp-192 (O2). This interaction is important for theconstruction of the model and the investigation of the enzymaticactivity. In the crystal structure, these two oxygens (O1-O2)are only 2.5 Å apart. Such a short distance between twoelectronegative atoms would require a large energetic penaltyif there were not a proton between the atoms. However, becauseour model is based on the 2.9 Å crystal structure, one can arguethat this short distance between the oxygens is a consequenceof insufficient accuracy in the solved structure. To answer thisquestion, we considered two scenarios. Assuming that there aresome inaccurately determined geometrical parameters in thecrystal structure, we first performed quantum chemical reopti-mization of the active site without a proton between the twooxygens. In optimization with this scheme, not only did the twooxygens come far apart from each other but also the wholemodel became dramatically distorted from the original crystalgeometry. On the basis of the results of the failed optimization,we assumed that protonation of one of these oxygen atoms mightbe necessary for the reproduction of the basic features of thecrystal structure. Indeed, quantum chemical optimization of theactive site with a proton between the two oxygen atoms leadsto an active site geometry that is very close to the experimentalone (see below). Thus, we conclude that the formation of ahydrogen bond between O2 of Asp-192 and O1 of theRphosphate is mandatory for the maintenance of the active sitegeometry.

In our model, the O1 atom of theR phosphate can beconsidered to be protonated. This makes the PR formally neutral.Accordingly, Asp-192 can be considered to be negativelycharged and interacts with two Mg, ions as is usually depictedin the schematic representation of the Polâ active site (Scheme1). The triphosphate substrate has a-3 total charge, similar tothe charge state in solution. However, in contrast to a uniformdistribution of the negative charges along a triphosphatecompound as found in solution, the enzymatic environmentshifts the negative electron density of the model triphosphatesubstrate toward theγ phosphate. This is due to the stronginteraction of the substrate with the Mg2 ion and the positivelycharged Arg-149 residue.

Figure 1. Optimized structure of the model for the Polâ active site.The experimental distances9 are in parentheses.

288 J. Phys. Chem. B, Vol. 105, No. 1, 2001 Abashkin et al.

To complete construction of the active site, a template primermodel fragment was added. Because only a very small portionof the template primer is directly involved in the catalyticprocess, we modeled part of the template primer as a hydroxylgroup that is attached to a methyl group. This further allowedus to keep the model computational tractable and allowedextensive searching of the stationary points on the multidimen-sional potential surface.

The overall charge of our model (Figure 1), consisting of 67atoms, is zero. Negative charges of compounds Asp-190 (-1),Asp-192 (-1), the substrate (-3), and the hydroxyl group (-1)coordinated to Mg1 are compensated by positive charges ontwo Mg ions (+4) and two Arg residues (+2).

The results of a constrained quantum chemical optimizationof our model show that the optimized geometry agrees verywell both qualitatively (Figure 2) and quantitatively (Figure 1)with the basic characteristics of the crystal active site. We stressthat only a very few restrictions were applied during theconstrained optimization. Namely, only the relative positionsof Arg-183, Arg-149, and Ser-180 were kept fixed. This wasachieved by fixing the positions of the methyl groups in theCH3(NH2)2

+ compounds modeling the arginine residues and themethyl moiety of the CH3OH compound modeling the serineresidue. Such restrictions allow free rotation of the functionalgroups for all three residues but prevent the residues frommoving with respect to each other. The substrate, template-primer model compound, and all components of two metalionsstwo aspartic residues of the principal catalytic fragmentwith the nearest environmentswere completely optimizedwithout any restrictions. Thus, the agreement between thegeometrical characteristics of the crystal and the model structureswere obtained not by forcing the model parameters to matchthe corresponding experimental data but by the nature of theconstructed active site.

Though good geometrical agreement between the experimen-tal and the optimized structure cannot unambiguously prove thatthe proposed model of the Polâ active site is the only onepossible, we are strongly encouraged by this excellent agreementand feel justified in using this model for the investigation ofthe enzymatic activity and possible reaction mechanisms ofPol â.

Results and Discussion

We investigated two potential mechanisms of Polâ. The firstmechanism which involves the formation of a pentacoordinatedintermediate was proposed on the basis of the crystallographicstudy of Polâ.9 A schematic representation of the reaction

mechanism is shown in Scheme 1. The basic premise of thisstepwise mechanism is the formation of the pentacoordinatedintermediate following a 3′-OH hydroxyl group attack on theR phosphate atom. The second step of the reaction involvescleavage of the PR-OPâ bond. Though it is impossible toestablish the validity of the proposed mechanism from acrystallographic structure, the observation that the pentacoor-dinated structure of the tentative intermediate fits well into thecrystal structure of Polâ and likely could be stabilized in theenzymatic environment9 suggests that this mechanism might bepossible.

The second mechanism that we have studied is a PO3

intermediate mechanism. Despite some arguments about thedetails, the involvement of a monomeric metaphosphate PO3

-

intermediate or metaphosphate-like transition state (TS) has beenobserved in some types of phosphate reactions in solution.22,23

We were interested in exploring whether the formation of a PO3

intermediate structure is favorable in the Polâ active site.Compared to the pentacoordinated mechanism, the PO3 inter-mediate reaction mechanism has a reverse order (Scheme 1).In the first step of the latter mechanism, the PR-OPâ bond iscleaved and the PO3 intermediate is formed. In the second step,the 3′-OH group attacks the PR atom, leading to the finalproducts.

To our knowledge, this mechanism has never been investi-gated for Polâ. However, this mechanism is worth consideringfrom an energetic standpoint because a hydroxyl group attackon a PO3-like compound is more likely than that on atetracoordinated phosphate. Indeed, the results of our modelcalculations show (Figure 3) that the energy needed foractivation of a phosphate oxidation process is 2 times smaller

Figure 2. Superposition of the optimized model structure (gray) andthe corresponding part of the crystal structure of ternary complexes ofrat DNA Pol â, a DNA template primer, and ddCTP (black).9

Figure 3. Comparison of the model reactions of an R-OH group attackon a PO3-like plane compound and the tetracoordinated phosphate.

Figure 4. TS structure leading to the PO3 intermediate.

Enzymatic Activity in DNA Polâ J. Phys. Chem. B, Vol. 105, No. 1, 2001289

in the case of the PO3 compound with respect to the PO4

compound case. These results are easy to interpret just consider-ing the electrostatic picture of the attack. In the case of the PO4

compound, the attacking oxygen atom has to overcome repulsionof three oxygen atoms en route to the more positive phosphateatom. In contrast, PO3 has almost a planar configuration with afavorable perpendicular direction of OH group attack on thephosphate atom. Thus, provided that the formation of a PO3

group in the active site of Polâ is feasible, the PO3 reaction

pathway might be an attractive alternative to the pentacoordi-nated channel.

PO3 Intermediate Mechanism.We started our investigationof the PO3 intermediate mechanism with a stepwise elongationof the PR-OPâ bond in the initial complex (Figure 1) andcontinual reoptimization of the corresponding structures. It wasassumed that in the process of breaking down the bond anadditional negative charge on theâ phosphate can be stabilizedby the nearby residue Arg-183 and by magnesium ions.

Figure 5. Reaction critical structures for the pentacoordinated reaction mechanism: (a) TS1 corresponding to the nucleophilic attack on the PRatom; (b) INT_I structure; (c) TSrot corresponding to the rotation about the PR-O2 bond; (d) INT_II structure; (e) TS2 structure resulting in thePR-OPâ bond cleavage; (f) final complex structure.

290 J. Phys. Chem. B, Vol. 105, No. 1, 2001 Abashkin et al.

However, following this reaction coordinate, we were not ableto find any TS structure because the energy of our modelconsistently rose and the PR-OPâ bond elongated (up to 4 Å).Thus, we concluded that electrostatic stabilization alone is notenough to permit the bond cleavage to occur.

Protonation of the oxygen in the course of the PR-OPâ bondcleavage might be an additional source of stabilization of thecorresponding TS. We found that the proton, which is sharedbetween the O1 atom of theR phosphate group and the O2atom of Asp-192 in the initial complex, shifts toward the Asp-192 oxygen while the PR-OPâ bond becomes longer. Such ashift is due to the changing nature of O1-PR from a single bond(with a negative charge on O1) to a double bond (with a neutralcharge on O1). The proton is now attached to Asp-192. Theprotonated Asp-192 can now move toward the oxygen of theâphosphate group and can serve as a source of a proton in thecourse of the PR-OPâ bond cleavage. In investigating thisscenario, we did find a TS that leads to the formation of thePO3 intermediate (Figure 4). Unfortunately, the activation energyfor this reaction step (37.0 kcal/mol) is too high to furtherconsider the formation of the intermediate as a feasible processin the model enzymatic environment. Thus, we failed to find areasonable pathway for the PO3 reaction mechanism.

Pentacoordinated Reaction Mechanism.The reaction criti-cal points of the pentacoordinated mechanism and the energeticreaction profile are shown in Figures 5 and 6, respectively. Thereaction starts from the initial complex depicted in Figure 1. Anucleophilic attack of the model hydroxyl compound on the PRatom occurs simultaneously with a proton transfer to the O2atom of theR phosphate group (Figure 5a). Our modeling ofthis reaction step shows that the Mg1 ion acts as a Lewis acidand polarizes the 3′-OH group of the primer strand. Suchpolarization facilitates deprotonation of the OH group and leadsto the creation of the attacking nucleophile. In our model, thenucleotide substrate itself plays the role of a general base andaccepts the proton from the incoming compound. Possibleinvolvement of Asp-256 in the process of the 3′-OH deproto-nation has been previously discussed.9 From our point of view,this is unlikely because Asp-256 is situated more than 5 Å apart

from the attacking nucleophile and the movement of thenegatively charged aspartic residue is restricted because of aninteraction with the Mg1 ion. In addition, such an interactiondecreases the ability of Asp-256 to be a proton acceptor. It isinteresting to note that, according to the results of experimentaland theoretical studies of triphosphate substrate hydrolysis inthe ras p21 protein environment, the substrate itself most likelyplays the role of general base and accepts a proton from anattacking water molecule.24,25 On the basis of the abovearguments, we believe that the TS1 structure shown in Figure5a corresponds to the most realistic model for the nucleophilicattack. The process needs approximately 13 kcal/mol to beactivated (Figure 6).

The nucleophilic attack results in the formation of thepentacoordinated intermediate INT_I (Figure 5b). It was foundthat INT_I is energetically less stable than the pentacoordinatedintermediate INT_II (Figure 5d) by 5.3 kcal/mol. The onlydifference between these two intermediates is the position ofthe proton that originally belonged to the attacking hydroxylcompound. In the INT_I structure, the proton is directed towardthe model primer compound, whereas in the INT_II structure,the proton is directed toward theâ and γ phosphate groups.The greater stability of INT_II can be rationalized by takinginto account the fact that a negative charge on the triphosphateis mainly localized onâ and γ phosphate groups. Therefore,the proton position in INT_II leads to the stabilization of theelectrostatic interaction between the proton and the negativesubstrate charge. The conformation INT_I can be converted tothe conformation INT_II through the rotational TS structure TSrot

(Figure 5c), with the barrier of 6.2 kcal/mol (Figure 6).On the final reaction stage, the cleavage of the PR-OPâ

bond in the INT_II structure occurs with a low activationenergy (5.7 kcal/mol). The corresponding TS structure is shownin Figure 5e. As seen from the figure, the proton transfer isneeded to break the phosphate bond. The final complex of theproduct and active site for the studied reaction is presented inFigure 5f.

Overall, in contrast to the PO3 reaction pathway, our resultsshow that the pentacoordinated mechanism is feasible in this

Figure 6. Reaction energetic profiles of the two investigated reaction pathways for nucleotide addition in Polâ: the PO3 intermediate mechanism,-dotted line; the pentacoordinated mechanism, dashed line.

Enzymatic Activity in DNA Polâ J. Phys. Chem. B, Vol. 105, No. 1, 2001291

model system. The rate-limiting step of the reaction is thenucleophilic attack, which needs 13 kcal/mol of activationenergy. We predict that the barrier of the corresponding TS,which is ionic, can be further lowered by taking into accountelectrostatic stabilization from the rest of the protein.

Conclusions

We investigated two different reaction pathways for nucle-otide addition in Polâ using DFT. Our model consists of 67atoms of the polymerase active site and includes all majorfeatures thought to be important for catalysis. Geometricaloptimization of our model active site resulted in a structure thatagrees very well both qualitatively and quantitatively with thecrystal structure. This agreement led us to believe that this modelis sufficient for our mechanism studies.

For the first time, we investigated the mechanism thatinvolves formation of a PO3 intermediate in Polâ. Thisintermediate is thought to be involved in phosphate reactionsin solution and could be accommodated in the Polâ active site.However, the barrier to formation of this intermediate is 37.0kcal/mol, and we do not expect that this mechanism is the onethat occurs in the enzyme. The second mechanism that leads toa pentacoordinated intermediate appears to be feasible. Ourcalculations are the first to characterize all reaction criticalstructures and to yield the whole energetic profile for thepentacoordinated mechanism. This stepwise mechanism hasrelatively low barriers and, after the nucleophilic attack, everystep of the reaction is exothermic.

Using the constructed model for the Polâ active site, it nowbecomes possible to characterize the individual contributionsof various active-site residues and of the two metals to thecatalytic mechanism. Such calculations are now in progress.

Acknowledgment. The authors thank Dr. Jack R. Collinsfor helpful discussions. This project has been funded in wholeor in part with Federal funds from the National Cancer Instituteand National Institute of Health, under Contract No. NO1-CO-56000. The content of this publication does not necessarilyreflect the views or policies of the Department of Health andHuman Services, nor does the mention of trade names, com-

mercial products, or an organization imply endorsement by theU.S. Government.

References and Notes

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