Polymerase-Tailored Variations in the Water-Mediated and Substrate-Assisted Mechanism for Nucleotidyl Transfer: Insights from a Study of T7 DNA Polymerase (original) (raw)
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Proceedings of the National Academy of Sciences, 2007
The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3-hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3-OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.
Journal of the American Chemical Society, 2013
DNA polymerase β (pol β) is a bifunctional enzyme widely studied for its roles in base excision DNA repair where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol β in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3′ group, thus activating O3′ as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1,000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wildtype enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3′ proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3′ proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3′. The theoretical results with the wild-type enzyme indicate early charge reorganization associated with the O3′ proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.
Nucleic acid polymerases use a general acid for nucleotidyl transfer
Nature Structural & Molecular Biology, 2009
Nucleic acid polymerases catalyze the formation of DNA or RNA from nucleoside-triphosphate precursors. Amino acid residues in the active site of polymerases are thought to contribute only indirectly to catalysis by serving as ligands for the two divalent cations that are required for activity or substrate binding. Two proton-transfer reactions are necessary for polymerase-catalyzed nucleotidyl transfer: deprotonation of the 3¢-hydroxyl nucleophile and protonation of the pyrophosphate leaving group. Using model enzymes representing all four classes of nucleic acid polymerases, we show that the proton donor to pyrophosphate is an active-site amino acid residue. The use of general acid catalysis by polymerases extends the mechanism of nucleotidyl transfer beyond that of the well-established two-metal-ion mechanism. The existence of an active-site residue that regulates polymerase catalysis may permit manipulation of viral polymerase replication speed and/or fidelity for virus attenuation and vaccine development.
Science China Chemistry, 2012
The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods. Molecular dynamics (MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential (GSBP) by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex were constructed based on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Three pathways were explored using the DFT method. The most favorable reaction pathway involves indirect proton migration from the RNA primer 3′-OH to the oxygen atom of -phosphate via a solvent water molecule, proton rotation from the oxygen atom of -phosphate to the -phosphate side, the RNA primer 3′-O nucleophilic attack on the -phosphorus atom, and P-O bond breakage. The corresponding reaction potential profile was obtained. The rate limiting step, with a barrier height of 21.5 kcal/mol, is the RNA primer 3′-O nucleophilic attack, rather than the commonly considered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for further studies. yeast RNA polymerase II, nucleotidyl transfer reaction, two-metal-ion mechanism, two-proton-transfer, active center
Nucleic Acids Research, 2012
Human DNA Pol i is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM-MD (Quantum Mechanics/ Molecular Mechanics-Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol i and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol i as well, suggesting its broad applicability. The hydrogen on the 3 0-OH primer terminus is transferred through crystal and solvent waters to the c-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the c-phosphate to the a,b-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene-derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol i.
Biochemistry, 2007
DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the ,γ-bridging O with CH 2 , CHF, CF 2 , or CCl 2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol). T‚G mismatches occur with fidelities similar to dGTP with the exception of the CH 2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with K d s between 1 and 4 µM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brønsted correlations of log(k pol) versus leaving-group pK a for both right and wrong base incorporation reveal similar sensitivities (lg ≈-0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pK a s of CF 2 , O, and the sterically bulky CCl 2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3′-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.
Journal of the American Chemical Society, 2017
DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1-3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PPi) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase β (hPolβ) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolβ in an open conformation with PPi bound in the act...
Computational delineation of the catalytic step of a high-fidelity DNA polymerase
Protein Science, 2010
The Bacillus fragment, belonging to a class of high-fidelity polymerases, demonstrates high processivity (adding~115 bases per DNA binding event) and exceptional accuracy (1 error in 10 6 nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two-metal-ion mechanism, and requiring the proton on the terminal primer O3 0 to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of~12 kcal/ mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.
Journal of Biological Chemistry, 2004
Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol. These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.
Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms
Theoretical Chemistry Accounts, 2012
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 b 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, substrateinduced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual ratedetermining steps for chemistry. Keywords Enzyme catalysis Á Intrinsic protein dynamics Á Pre-chemistry conformational adjustments Á Nucleotidyl transfer Á DNA polymerase b Á Catalytic cycle chemical step