Conformational transition pathway of polymerase beta/DNA upon binding correct incoming substrate

The closing conformational transition of wild-type polymerase beta bound to DNA template/primer before the chemical step (nucleotidyl transfer reaction) is simulated using the stochastic difference equation (in length version, "SDEL") algorithm that approximates long-time dynamics. The order of the events and the intermediate states during pol beta's closing pathway are identified and compared to a separate study of pol beta using transition path sampling (TPS) (Radhakrishnan, R.; Schlick, T. Proc. Natl. Acad. Sci. USA 2004, 101, 5970-5975). Results highlight the cooperative and subtle conformational changes in the pol beta active site upon binding the correct substrate that may help explain DNA replication and repair fidelity. These changes involve key residues that differentiate the open from the closed conformation (Asp192, Arg258, Phe272), as well as residues contacting the DNA template/primer strand near the active site (Tyr271, Arg283, Thr292, Tyr296) and residues contacting the beta and gamma phosphates of the incoming nucleotide (Ser180, Arg183, Gly189). This study compliments experimental observations by providing detailed atomistic views of the intermediates along the polymerase closing pathway and by suggesting additional key residues that regulate events prior to or during the chemical reaction. We also show general agreement between two sampling methods (the stochastic difference equation and transition path sampling) and identify methodological challenges involved in the former method relevant to large-scale biomolecular applications. Specifically, SDEL is very quick relative to TPS for obtaining an approximate path of medium resolution and providing qualitative information on the sequence of events; however, associated free energies are likely very costly to obtain because this will require both successful further refinement of the path segments close to the bottlenecks and large computational time.     

Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms

In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.     

Unfavorable electrostatic and steric interactions in DNA polymerase β E295K mutant interfere with the enzyme's pathway

Mutations in DNA polymerase β (pol β) have been associated with approximately 30% of human tumors. The E295K mutation of pol β has been linked to gastric carcinoma via interference with base excision repair. To interpret the different behavior of E295K as compared to wild-type pol β in atomic and energetic detail, we resolve a binary crystal complex of E295K at 2.5 ? and apply transition path sampling (TPS) to delineate the closing pathway of the E295K pol β mutant. Conformational changes are important components in the enzymatic pathway that lead to and ready the enzyme for the chemical reaction. Our analyses show that the closing pathway of E295K mutant differs from the wild-type pol β in terms of the individual transition states along the pathway, associated energies, and the active site conformation in the final closed form of the mutant. In particular, the closed state of E295K has a more distorted active site than the active site in the wild-type pol β. In addition, the total energy barrier in the conformational closing pathway is 65 ± 11 kJ/mol, much higher than that estimated for both correct (e.g., G:C) and incorrect (e.g., G:A) wild-type pol β systems (42 ± 8 and 45 ± 7 kJ/mol, respectively). In particular, the rotation of Arg258 is the rate-limiting step in the conformational pathway of E295K due to unfavorable electrostatic and steric interactions. The distorted active site in the closed relative to open state and the high energy barrier in the conformational pathway may explain in part why the E295K mutant is observed to be inactive. Interestingly, however, following the closing of the thumb but prior to the rotation of Arg258, the E295K mutant complex has a similar energy level as compared to the wild-type pol β. This suggests that the E295K mutant may associate with DNA with similar affinity, but it may be hampered in continuing the process of chemistry. Supporting experimental data come from the observation that the catalytic activity of wild-type pol β is hampered when E295K is present: this may arise from the competition between E295K and wild-type enzyme for the DNA. These combined results suggest that the low insertion efficiency of E295K mutant as compared to wild-type pol β may be related to a closed form distorted by unfavorable electrostatic and steric interactions between Arg258 and other key residues. The active site is thus less competent for proceeding to the chemical reaction, which may also involve a higher reaction barrier than the wild-type or may not be possible in this mutant. Our analysis also suggests further experiments for other mutants to test the above hypothesis and dissect the roles of steric and electrostatic factors on enzyme behavior.     

Fidelity discrimination in DNA polymerase beta: differing closing profiles for a mismatched (G:A) versus matched (G:C) base pair

Understanding fidelity-the faithful replication or repair of DNA by polymerases-requires tracking of the structural and energetic changes involved, including the elusive transient intermediates, for nucleotide incorporation at the template/primer DNA junction. We report, using path sampling simulations and a reaction network model, strikingly different transition states in DNA polymerase beta's conformational closing for correct dCTP versus incorrect dATP incoming nucleotide opposite a template G. The cascade of transition states leads to differing active-site assembly processes toward the "two-metal-ion catalysis" geometry. We demonstrate that these context-specific pathways imply different selection processes: while active-site assembly occurs more rapidly with the correct nucleotide and leads to primer extension, the enzyme remains open longer, has a more transient closed state, and forms product more slowly when an incorrect nucleotide is present. Our results also suggest that the rate-limiting step in pol beta's conformational closing is not identical to that for overall nucleotide insertion and that the rate-limiting step in the overall nucleotide incorporation process for matched as well as mismatched systems occurs after the closing conformational change.     

Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization

The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3'H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-P(alpha) then migrates to the gamma-phosphate oxygen of the incoming nucleotide as O3' attacks P(alpha), and the P(alpha)-O3A bond breaks. The other possible pathway in which the O3'H proton is transferred directly to O1A on P(alpha) has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for P(alpha) occurs during the concerted bond formation (O3'-P(alpha)) and breaking (P(alpha)-O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3'H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase beta from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs 15.0 kcal/mol) due to the more solvent-exposed active site.     

Single-molecule Förster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I

Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule F?rster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair.     

DNA polymerase beta catalysis: are different mechanisms possible?

DNA polymerases are crucial constituents of the complex cellular machinery for replicating and repairing DNA. Discerning mechanistic pathways of DNA polymerase on the atomic level is important for revealing the origin of fidelity discrimination. Mammalian DNA polymerase beta (pol beta), a small (39 kDa) member of the X-family, represents an excellent model system to investigate polymerase mechanisms. Here, we explore several feasible low-energy pathways of the nucleotide transfer reaction of pol beta for correct (according to Watson-Crick hydrogen bonding) G:C basepairing versus the incorrect G:G case within a consistent theoretical framework. We use mixed quantum mechanics/molecular mechanics (QM/MM) techniques in a constrained energy minimization protocol to effectively model not only the reactive core but also the influence of the rest of the enzymatic environment and explicit solvent on the reaction. The postulated pathways involve initial proton abstraction from the terminal DNA primer O3'H group, nucleophilic attack that extends the DNA primer chain, and elimination of pyrophosphate. In particular, we analyze several possible routes for the initial deprotonation step: (i) direct transfer to a phosphate oxygen O(Palpha) of the incoming nucleotide, (ii) direct transfer to an active site Asp group, and (iii) transfer to explicit water molecules. We find that the most probable initial step corresponds to step (iii), involving initial deprotonation to water, which is followed by proton migration to active site Asp residues, and finally to the leaving pyrophosphate group, with an activation energy of about 15 kcal/mol. We argue that initial deprotonation steps (i) and (ii) are less likely as they are at least 7 and 11 kcal/mol, respectively, higher in energy. Overall, the rate-determining step for both the correct and the incorrect nucleotide cases is the initial deprotonation in concert with nucleophilic attack at the phosphate center; however, the activation energy we obtain for the mismatched G:G case is 5 kcal/mol higher than that of the matched G:C complex, due to active site structural distortions. Taken together, our results support other reported mechanisms and help define a framework for interpreting nucleotide specificity differences across polymerase families, in terms of the concept of active site preorganization or the so-called "pre-chemistry avenue".     

Computer simulation of the chemical catalysis of DNA polymerases: discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase

Understanding the chemical step in the catalytic reaction of DNA polymerases is essential for elucidating the molecular basis of the fidelity of DNA replication. The present work evaluates the free energy surface for the nucleotide transfer reaction of T7 polymerase by free energy perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect of the enzyme simulation is a comparison of enzymatic free energy profiles with the corresponding reference reactions in water using the same computational methodology, thereby enabling a quantitative estimate for the free energy of the nucleotide insertion reaction. The reaction is driven by the FEP/EVB methodology between valence bond structures representing the reactant, pentacovalent intermediate, and the product states. This pathway corresponds to three microscopic chemical steps, deprotonation of the attacking group, a nucleophilic attack on the P(alpha) atom of the dNTP substrate, and departure of the leaving group. Three different mechanisms for the first microscopic step, the generation of the RO(-) nucleophile from the 3'-OH hydroxyl of the primer, are examined: (i) proton transfer to the bulk solvent, (ii) proton transfer to one of the ionic oxygens of the P(alpha) phosphate group, and (iii) proton transfer to the ionized Asp654 residue. The most favorable reaction mechanism in T7 pol is predicted to involve the proton transfer to Asp654. This finding sheds light on the long standing issue of the actual role of conserved aspartates. The structural preorganization that helps to catalyze the reaction is also considered and analyzed. The overall calculated mechanism consists of three subsequent steps with a similar activation free energy of about 12 kcal/mol. The similarity of the activation barriers of the three microscopic chemical steps indicates that the T7 polymerase may select against the incorrect dNTP substrate by raising any of these barriers. The relative height of these barriers comparing right and wrong dNTP substrates should therefore be a primary focus of future computational studies of the fidelity of DNA polymerases.     

Effect of Mn2+, Co2+, Ni2+, and Cu2+ on horseradish peroxidase: activation,inhibition, and denaturation studies

The effects of transition metal ions (M2+) such as Mn2+, Co2+, Ni2+, and Cu2+ on the functional and structural stabilities of horseradish peroxidase (HRP) were investigated with respect to reversible chemical denaturation, Michaelis-Menten kinetics, chemical modification and time-dependent catalytic activity. Conformational Gibbs free energy (deltaGo(H2O)) as a structural stability criterion and transition concentrations of metal ions ([M2+] 1/2) were estimated using a two-state chemical denaturation model. Activation and inhibitory concentration ranges for each metal ion were specified by the steady-state enzyme kinetics. Results of a pH-profile method confirmed by chemical modification indicate that a histidine residue interacts in the activation concentration range, whereas carboxylic residues (Asp and Glu) contribute to interaction in the inhibitory concentration range. Incubation of the enzyme with the metal ion at activation concentration leads to long-term functional stability of peroxidase. Thus, such metal ions as potent effectors induced the enhancement of conformational and functional stabilities of horseradish peroxidase.     

Hybrid QM/MM and DFT investigations of the catalytic mechanism and inhibition of the dinuclear zinc metallo-beta-lactamase CcrA from Bacteroides fragilis

Based on hybrid QM/MM molecular dynamics simulation and density functional theoretical (DFT) calculations, we investigate the mechanistic and energetic features of the catalytic action of dizinc metallo-beta-lactamase CcrA from Bacteroides fragilis. The 200 ps QM/MM simulation of the CcrA enzyme in complex with nitrocefin shows that the substrate beta-lactam moiety is directed toward the active site dizinc center through the interactions of aminocarbonyl and carboxylate groups with the two active site zinc ions and the two conserved residues, Lys167 and Asn176. From the determination of the potential energy profile of a relevant enzymatic reaction model, it is found that the nucleophilic displacement reaction step proceeds with a low-barrier height, leading to the formation of an energetically favored reaction intermediate. The results also show that the high catalytic activity of the CcrA enzyme stems from a simultaneous operation of three catalytic components: activation of the bridging hydroxide nucleophile by zinc-coordinated Asp86; polarization of the substrate aminocarbonyl group by the first zinc ion; stabilization of the negative charge developed on the departing amide nitrogen by the second zinc ion. Consistent with the previous experimental finding that the proton-transfer reaction step is rate-limiting, the activation energy of the second step is found to be 1.6 kcal/mol higher than that of the first step. Finally, through an examination of the structural and energetic features of binding of a thiazolidinecarboxylic acid inhibitor to the active site dizinc center, a two-step inhibition mechanism involving a protonation-induced ligand exchange reaction is proposed for the inhibitory action of a tight-binding inhibitor possessing a thiol group.     

Implications of active site constraints on varied DNA polymerase selectivity

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.     

Improving metal ion specificity during in vitro selection of catalytic DNA

Metal ions play important roles in both the structure and function of catalytic DNA and RNA. While most natural catalytic RNA molecules (ribozymes) are active in solutions containing Mg(2+), in vitro selection makes it possible to search for new catalytic DNA/RNA that are specific for other metal ions. However, previous studies have indicated that the in vitro selection protocols often resulted in catalytic DNA/RNA that were equally active or sometimes even more active with metal ions other than the metal ion of choice. To improve the metal ion specificity during the in vitro selection process, we implemented a negative selection strategy where the nucleic acid pool was subjected to a solution containing competing metal ions. As a result, those nucleic acids that were active with those metal ions are discarded. To demonstrate the effectiveness of the negative selection strategy, we carried out two parallel in vitro selections of Co(2+)-dependent catalytic DNA. When no negative selection was used in the selection process, the resulting catalytic DNA molecules were more active in solutions of Zn(2+) and Pb(2+) than in Co(2+). On the other hand, when the negative selection steps were inserted between the normal positive selection steps, the resulting catalytic DNA molecules were much more active with Co(2+) than in other metal ions including Zn(2+) and Pb(2+). These results suggest strongly that in vitro selection can be used to obtain highly active and specific transition metal ion-dependent catalytic DNA/RNA, which hold great promise as versatile and efficient endonucleases as well as sensitive and selective metal ion sensors.     

Computational modeling of the catalytic mechanism of human placental alkaline phosphatase (PLAP)

Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. Quantum mechanical, molecular dynamics, and molecular docking techniques were applied to computationally model the catalytic mechanism of human placental AP (PLAP). Kinetic and thermodynamic evaluations were performed for each reaction step. The functional significances of the more important residues within the active site were analyzed. The role of the metal ion at the metal binding site M3 was also examined. The calculated activation and reaction energy and free energy values obtained suggested the nucleophilic attack of the Ser92 alkoxide on the phosphorus atom of the substrate would be the rate-limiting step of the catalytic hydrolysis of alkyl phosphate monoesters by PLAP. The reactivities of the wild-type M3-Mg enzyme and the M3-Zn protein were compared, and the main difference observed was a change in the coordination number of the M3 metal for the M3-Zn enzyme. This modification in the active site structure lowered the free energy profile for the second chemical step of the catalytic mechanism (hydrolysis of the covalent phosphoserine intermediate). Consequently, a greater stabilization of the phosphoseryl moiety resulted in a small increment in the activation free energy of the phosphoserine hydrolysis reaction. These computational results suggest that the activation of APs by magnesium at the M3 site is caused by the preference of Mg(2+) for octahedral coordination, which structurally stabilizes the active site into a catalytically most active conformation. The present theoretical results are in good agreement with previously reported experimental studies.     

Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.     

A quantum mechanical investigation of possible mechanisms for the nucleotidyl transfer reaction catalyzed by DNA polymerase beta

Several quantum mechanical (QM) and hybrid quantum/molecular mechanical (QM/MM) studies have been employed recently to analyze the nucleotidyl transfer reaction in DNA polymerase beta (pol beta). Our examination reveals strong dependence of the reported mechanism on the initial molecular model. Thus, we explore here several model systems by QM methods to investigate pol beta's possible pathway variations. Although our most favorable pathway involves a direct proton transfer from O3'(primer) to O2alpha(Palpha), we also discuss other initial proton-transfer steps--to an adjacent water, to triphosphate, or to aspartic units--and the stabilizing effect of crystallographic water molecules in the active site. Our favored reaction route has an energetically undemanding initial step of less than 1.0 kcal/mol (at the B3LYP/6-31G(d,p) level), and involves a slight rearrangement in the geometry of the active site. This is followed by two major steps: (1) direct proton transfer from O3'(primer) to O2alpha(Palpha) leading to the formation of a pentavalent, trigonal bipyramidal Palpha center, via an associative mechanism, at a cost of about 28 kcal/mol, and (2) breakage of the triphosphate unit (exothermic process, approximately 22 kcal/mol) that results in the full transfer of the nucleotide to the DNA and the formation of pyrophosphate. These energy values are expected to be lower in the physical system when full protein effects are incorporated. We also discuss variations from this dominant pathway, and their impact on the overall repair process. Our calculated barrier for the chemical reaction clearly indicates that chemistry is rate-limiting overall for correct nucleotide insertion in pol beta, in accord with other studies. Protonation studies on relevant intermediates suggest that, although protonation at a single aspartic residue may occur, the addition of a second proton to the system significantly disturbs the active site. We conclude that the active site rearrangement step necessary to attain a reaction-competent geometry is essential and closely related to the "pre-chemistry" avenue described recently as a key step in the overall kinetic cycle of DNA polymerases. Thus, our work emphasizes the many possible ways for DNA polymerase beta's chemical reaction to occur, determined by the active site environment and initial models.     

Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.     

Hydrophobicity, shape, and pi-electron contributions during translesion DNA synthesis

Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template, represents a common route toward mutagenesis and possibly disease development. To further define the mechanism of this promutagenic process, we synthesized and tested the enzymatic incorporation of two isosteric 5-substituted indolyl-2'deoxyriboside triphosphates opposite an abasic site. The catalytic efficiency for the incorporation of the 5-cyclohexene-indole derivative opposite an abasic site is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects a substantial increase in the k(pol) value (compare 25 versus 0.5 s(-1), respectively) as opposed to an influence on ground-state binding of either non-natural nucleotide. The faster k(pol) value for the 5-cyclohexene-indole derivative indicates that pi-electron density enhances the rate of the enzymatic conformational change step required for insertion opposite the abasic site. However, the kinetic dissociation constants for the non-natural nucleotides are identical and indicate that pi-electron density does not directly influence ground-state binding opposite the DNA lesion. Surprisingly, each non-natural nucleotide can be incorporated opposite natural templating bases, albeit with a greatly reduced catalytic efficiency. In this instance, the lower catalytic efficiency is caused by a substantial decrease in the k(pol) value rather than perturbations in ground-state binding. Collectively, these data indicate that the rate of the conformational change during translesion DNA synthesis depends on pi-electron density, while the enhancement in ground-state binding appears related to the size and shape of the non-natural nucleotide.     

argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli contains a dinuclear metalloactive site

The catalytic and structural properties of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by approximately 20 times. Moreover, ArgE exhibits approximately 90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-L-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 degrees C, indicating that the rate-limiting step does not change over this temperature range. The activation energy for NAO hydrolysis by ArgE was 25.6 kJ/mol when loaded with Zn(II) and 34.3 kJ/mol when loaded with Co(II). Electronic absorption and EPR (electron paramagnetic resonance) spectra of [Co x (ArgE)] and [CoCo(ArgE)] indicate that both divalent metal binding sites are five coordinate. In addition, EPR data show clear evidence of spin-spin coupling between the Co(II) ions in the active site but only after addition of a second equivalent of Co(II). Combination of these data provides the first physical evidence that the ArgE from E. coli contains a dinuclear Zn(II) active site, similar to AAP and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2).     

A triad interaction in the fingers subdomain of DNA polymerase beta controls polymerase activity

DNA polymerase beta (pol beta) is the main polymerase involved in the base excision repair pathway responsible for repairing damaged bases in the DNA. Previous studies on the H285D mutant of pol beta suggested that the C-terminal region of the polymerase is important for polymerase function. In this study, the C-terminal region of pol beta was mutated to assess its role in polymerization. Kinetic experiments showed that the C-terminal region is required for wild-type polymerase activity. Additionally, an interaction between the fingers and palm subdomain revealed itself to be required for polymerase activity. The E316R mutant of pol beta was shown to have a 29,000-fold reduction in polymerization rate with no reduction in nucleotide binding, suggesting that there exists a noncovalent mechanistic step between nucleotide binding and nucleophilic attack of the primer 3'-hydroxyl group on the α-PO(4) of the nucleotide. Molecular modeling studies of the E316R mutant demonstrate that disrupting the interaction between Arg182 and Glu316 disrupts the packing of side chains in the hydrophobic hinge region and may be hampering the conformational change during polymerization. Taken together, these data demonstrate that the triad interaction of Arg182, Glu316, and Arg333 is crucial for polymerase function.