Locality principle in wave mechanics

This paper proves a locality principle for a wave-mechanical particle governed by the Schrödinger equation. It is shown that ρ(r,β), the Laplace transform of the local density of states n(r,E), depends significantly only on the potential V(r′) at points r′ near r. The effect of changes of V(r′) at distant points r′ (|x_i′ - x_i| > a) on ρ(r,β) decay in a Gaussian fashion with a. This result sheds some light on the locality of physical properties of extended systems and provides general support for various local methods of calculation.     

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

(E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (K_m for Mg²⁺ ≈ 150 μM; K_m for Mn²⁺ ≈ 7 μM). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-β-farnesene (85%), (Z)-β-farnesene (8%), and δ-cadinene (5%) with the native C15 substrate farnesyl diphosphate (K_m ≈ 0.6 μM; V_rel = 100) and Mg²⁺ as cofactor, and (E)-β-farnesene (98%) and (Z)-β-farnesene (2%) with Mn²⁺ as cofactor (V_rel = 80). With the C₁₀ analog, GDP, as substrate (K_m = 1.5 μM; V_rel = 3 with Mg²⁺ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.     

Assembly and mechanism of a group II ECF transporter

Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A′), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A′ and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A–A′ heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.     

Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase

The Chlamydomonas reinhardtii chloroplast mutants 18-5B and 18-7G lack both the chloroplast-encoded large subunit and nuclear-encoded small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). A chloroplast intergenic-suppression model has been postulated to account for the genetic instability of 18-5B revertants. Here, we have determined the molecular basis of the 18-5B and 18-7G mutants. They contain nonsense mutations close to the 3′ and 5′ ends of their large-subunit genes, respectively. Pulse-chase experiments revealed that the 18-5B mutant produces a truncated large subunit that is unstable. In connection with previous experiments, this work identifies nonsense suppression in the chloroplast. Small subunits are also synthesized but then degraded in the mutants. Thus, the coordinated absence of subunits is achieved through degradation of the small subunit in the specific absence of the large subunit.     

Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids

It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of “unusual” e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. ¹⁸O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O₂-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.     

Average time until fixation of a mutant allele in a finite population under continued mutation pressure: Studies by analytical, numerical, and pseudo-sampling methods

We consider a single locus, and denote by A the wild-type allele and by A′ the mutant allele that is produced irreversibly in each generation from A at the rate v. Let 1 + s, 1 + h, and 1 be, respectively, the relative fitnesses of mutant homozygote A′A′, mutant heterozygote A′A, and wild-type homozygote AA. Then, it is shown, on the basis of the diffusion equation method, that the average time until fixation of the mutant allele (A′) in a randomly mating population of effective size N_e, given that the initial frequency is p, is [Formula: see text] in which B(x) = (S/2)x² + Hx(1 - x), S = 4N_es, H = 4N_eh, and V = 4N_ev. Of particular interest are the cases in which the mutant allele is deleterious (s = -s′, s′ > 0). Three cases are considered; the mutant is: (i) completely dominant s = h = -s′, (ii) completely recessive s = -s′, h = 0, and (iii) semidominant s = -s′, h = -s′/2, in which s′ is the selection coefficient against the mutant homozygote. It is shown that the average time until fixation is shorter when the deleterious mutant allele is dominant than when it is recessive if 4N_ev is larger than 1. On the other hand, the situation is reversed if 4N_ev is smaller than 1. It is also shown that for a mutant allele for which N_es′ > 10, it takes such a long time until fixation that we can practically ignore the occurrence of random fixation of a deleterious allele under continued mutation pressure. To supplement the analytical treatment, extensive simulation experiments were performed by using a device called the pseudo-sampling variable, which can enormously accelerate the process of simulation by a computer. This method simulates the diffusion process itself rather than the binominal sampling process (in population genetics the diffusion model is usually regarded as an approximation of the discrete binomial sampling process).     

Specific glycosphingolipids mediate epithelial-to-mesenchymal transition of human and mouse epithelial cell lines

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with d-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-β, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-β is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-β treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-β-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).     

A single historical substitution drives an increase in acetylcholine receptor complexity

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral β-subunit can not only replace the extant β-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral β-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.

The muscle-type acetylcholine receptor (AChR) is the prototypical ligand-gated ion channel, which evolved to convert chemical signals into electrical impulses in order to initiate muscle contraction (1). The adult form of the human AChR contains five related subunits (1), which are the products of four paralogous genes (2). The subunits assemble with a precise stoichiometry and arrangement to form a heteropentameric barrel-shaped structure around a central ion-conducting pore (Fig. 1A; counterclockwise α-ε-α-δ-β) (3–7). The presence of multiple differentiated AChR subunits implies their functional specialization. Indeed, agonist recognition occurs at two chemically distinct binding sites located at the interface between the α-subunits and their neighboring δ- or ε-subunit (5, 8, 9). The chemical inequivalence of the two agonist-binding sites illustrates the structural complexity of the heteromeric complex, as well as the apparent functional fine-tuning that resulted from subunit diversification and specialization during eons of evolution.Open in a separate windowFig. 1.Electrical fingerprinting reveals the number of β_Anc subunits in β_Anc-containing AChRs. (A) The subunit stoichiometry and arrangement of wild-type human adult AChRs is well established (Top), whereas it is unknown for β_Anc-containing AChRs (Bottom). (B) Representative single-channel bursts from cells cotransfected with cDNAs encoding wild-type human α-, δ-, and ε-subunits and either a HC mutant β_Anc [Top; β_Anc(T2′G), purple circle with black dot] or LC β_Anc (Middle; purple circle) or a mixture of both HC and LC β_Anc cDNAs (Bottom). The bursts shown were derived from a single patch in each of the HC, LC, and HC+LC experiments and were recorded at −120 mV in the presence of 30 μM acetylcholine. Bar graphs to the Right with dashed sight lines depict the mean amplitude of each observable class. The scale bar on the Bottom Right represents 50 ms and 10 pA. (C) Event-based amplitude histograms from the corresponding conditions in B reveal the number of amplitude classes resolved in each case. See SI Appendix, Table S2, for the number of bursts and patches included in each histogram.To gain insight into this evolutionary process and uncover cryptic AChR structure–function relationships, we have begun to reconstruct ancestral AChR subunits. Based on an original molecular phylogeny, we reconstructed the sequence of a putative ancestral AChR β-subunit, named β_Anc (10). This synthetic β-subunit differed by 132 amino acids from its human counterpart but was able to substitute for the human β-subunit in terms of both cell surface expression and function. β_Anc-containing hybrid AChRs had reduced conductance relative to wild type, and attempts to identify the origins of this difference uncovered an unappreciated dependence between historical substitutions within the AChR pore (11). The contribution of individual substitutions to AChR conductance depended upon the order in which they occurred as well as the background in which they were installed, demonstrating how the function of modern receptors is contingent upon evolutionary history. Although the mechanistic basis for this contingency was unclear, the timing of one of the substitutions hinted that it could have been involved in AChR subunit diversification and thus that the contingency might be a consequence of the emergence of a heteropentameric structure.Here, using concatenated subunits and a single-channel electrical fingerprinting strategy, we show that β_Anc is able to substitute for the human β-subunit and also supplant the human δ-subunit, rendering it obsolete. Remarkably, the ability of β_Anc to functionally replace the δ-subunit can be abolished by a single substitution that occurred early in the evolutionary history of the muscle-type β-subunit. Our finding that a historical substitution alters the subunit composition of functional AChRs provides an experimental demonstration of how a single mutation can necessitate an increase in AChR structural complexity and thus drive AChR subunit entrenchment. In addition, because the effect of this same substitution is contingent upon the background in which it occurs (11), our findings provide a mechanistic link between subunit entrenchment and historical contingency in this prototypical ligand-gated ion channel.     

Local density functional theory of atoms and molecules

A local density functional theory of the ground electronic states of atoms and molecules is generated from three assumptions: (i) The energy functional is local. (ii) The chemical potential of a neutral atom is zero. (iii) The energy of a neutral atom of atomic number Z is -0.6127 Z^7/3. The energy functional is shown to have the form [Formula: see text] where A₀=6.4563 and B₀=1.0058. The first term represents the electronic kinetic energy, the second term represents the electron—electron repulsion energy for N electrons, and the third term is the nucleus—electron attraction energy. The energy E and the electron density ρ are obtained and discussed in detail for atoms; their general properties are described for molecules. For any system the density becomes zero continuously at a finite distance from nuclei, and contours of the density are contours of the bare-nuclear potential v. For an atomic species of fractional charge q = 1 - (N/Z), an energy formula is obtained, [Formula: see text] which fits Hartree—Fock energies of 625 atoms and ions with root-mean-square error of 0.0270. A more general local density functional involving a coefficient B(N) = B₀N^2/3 + B₁ is briefly considered.     

A Xenopus oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit

Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca²⁺ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α₁ cRNAs (α_1C and α_1E); (ii) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α₁ Ca²⁺ channel currents; and (iii) coinjection of mammalian α₁ cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca²⁺ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α₁ alone” channels are in fact tightly associated α₁β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α₁β]β_n complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α₁β, are [α₁β]β_n complexes that associate with α₂δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α_1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α₁ subunits.     

Primary electron transfer reactions in modified reaction centers from Rhodopseudomonas sphaeroides

Absorption spectra were measured by means of an optical multichannel analyzer in Rhodopseudomonas sphaeroides R-26 reaction centers (RCs) modified by treatment with NaBH₄ at various times (≥1 ps) after the onset of a short excitation flash at 880 nm. Most of these RCs (75-95%) have only one “monomeric” bacteriochlorophyll-800 (B₁) molecule and are as active as the original RCs. The duration of the excitation and measuring pulses was ≈33 ps. If the center of the excitation pulse preceded the center of the measuring pulse by 36-40 ps, the formation of a state P^E (early state), which is converted to the state P^F (P⁺ bacteriopheophytin^-) in 4 ± 1 ps (1/e time), was observed. Also the kinetics and the spectrum of the stimulated emission (reflecting the kinetics and the emission spectrum of the excited state P^*) were determined. The difference spectrum of the state P^E approximately equals the sum of the spectra of the states P^* (≈65%) and ¹[P⁺B₁^-] (≈35%). This indicates that B₁^- is an intermediate in the electron transfer from P^* to bacteriopheophytin, H₁, transferring this electron with a rate constant of (4 × 0.35 ps)^-1 = 7 × 10¹¹ s^-1.     

From the Cover: Intake and transformation to a glycoside of (Z)-3-hexenol from infested neighbors reveals a mode of plant odor reception and defense

Plants receive volatile compounds emitted by neighboring plants that are infested by herbivores, and consequently the receiver plants begin to defend against forthcoming herbivory. However, to date, how plants receive volatiles and, consequently, how they fortify their defenses, is largely unknown. In this study, we found that undamaged tomato plants exposed to volatiles emitted by conspecifics infested with common cutworms (exposed plants) became more defensive against the larvae than those exposed to volatiles from uninfested conspecifics (control plants) in a constant airflow system under laboratory conditions. Comprehensive metabolite analyses showed that only the amount of (Z)-3-hexenylvicianoside (HexVic) was higher in exposed than control plants. This compound negatively affected the performance of common cutworms when added to an artificial diet. The aglycon of HexVic, (Z)-3-hexenol, was obtained from neighboring infested plants via the air. The amount of jasmonates (JAs) was not higher in exposed plants, and HexVic biosynthesis was independent of JA signaling. The use of (Z)-3-hexenol from neighboring damaged conspecifics for HexVic biosynthesis in exposed plants was also observed in an experimental field, indicating that (Z)-3-hexenol intake occurred even under fluctuating environmental conditions. Specific use of airborne (Z)-3-hexenol to form HexVic in undamaged tomato plants reveals a previously unidentified mechanism of plant defense.In response to herbivory, plants emit specific blends of volatiles (1). When undamaged plants are exposed to volatiles from neighboring herbivore-infested plants, they begin to defend against the impending infestation of herbivores (2, 3). This so-called “plant–plant signaling” has been reported in several plant species (4). For example, a study on the expression profiles of defense-related genes when Arabidopsis was exposed to several volatiles, including green leaf volatiles and a monoterpene, showed that the manner of induction varied with the gene monitored or the volatile used, suggesting that the plant responses were specific to the individual volatile compound (5). Kost and Heil (6) reported that the secretion of extrafloral nectar (an alternative food for carnivores) in undamaged lima bean plants was enhanced by volatiles from infested conspecific plants; this reaction was specific to (Z)-3-hexenyl acetate. Recently, Kikuta et al. (7) showed that wound-induced volatile organic compounds from Chrysanthemum cinerariaefolium induced the biosynthesis of pyrethrins in volatile-exposed neighboring plants. In this plant–plant signaling system, a blend of five compounds at specific concentrations was essential for the pyrethrin biosynthesis in receiver plants.These previous studies on plant–plant signaling raise questions about how different airborne volatiles are received by undamaged neighboring plants. Tamogami et al. (8) reported that airborne (E)-nerolidol was metabolized by Achyranthes bidentata plants into (E)-4,8-dimethyl-1,3,7-nonatriene. However, the mechanisms involved in the reception of airborne (E)-nerolidol in plants remained unclear. To date, only the receptor for ethylene, ETR1, a typical histidine kinase involved in a two-component regulatory system, has been identified (9, 10); no information exists on receptors for other volatile compounds in plants. In this study, we conducted comprehensive analyses of metabolic changes in intact tomato plants (Solanum lycopersicum) exposed to volatiles emitted from conspecifics infested with common cutworm (CCW; Spodoptera litura) and also conducted bioassays and biochemical analyses. We report that (Z)-3-hexenol emitted from herbivore-infested tomato plants is used by undamaged plants to form a glycoside with defensive function against CCW.     

Action of Rifamycin Derivatives on RNA Polymerase of Human Leukemic Lymphocytes

The mechanism of inhibition of human RNA polymerase by four rifamycin derivatives was investigated. Derivative AF/013 (3-formyl rifamycin SV:O-n-octyloxime) with strong hydrophobic side chains prevents the polymerase from binding to DNA and also affects the size of RNA synthesized. Derivative PR/19 (3′-acetyl-1′-benzyl-2′-methylpyrrolo[3,2-c]-4-desoxy-rifamycin SV) only affects RNA synthesis when RNA polymerase has been previously incubated with the drug or when the reaction was performed at high salt concentration [0.14 M (NH₄)₂-SO₄]. Our results suggest that these drugs exert their inhibitory actions by binding to the enzyme instead of DNA.     

RNA-Dependent DNA Polymerase: Possible Role in the Amplification of Ribosomal DNA in Xenopus oocytes

2′,5′ - dimethyl - N(4′)benzyl - N(4′) - [desmethyl]rifampicin, a derivative of rifampicin, is used to acquire autoradiographic evidence that RNA-dependent DNA synthesis is involved in gene amplification during the early oogenesis of Xenopus laevis.     

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b₅, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This ω-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.     

Multiple deprotonation paths of the nucleophile 3′-OH in the DNA synthesis reaction

DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3′-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3′-OH in the reaction catalyzed by human DNA polymerase (Pol) η and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3′-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3′-OH using the 2′-OH of a ribonucleotide or 2′-F has little effect on catalysis. Moreover, combining the S113A mutation with 2′-F replacement, which removes two potential hydrogen acceptors of the 3′-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3′ via alternative paths, supporting the hypothesis that binding of the third Mg²⁺ initiates the reaction by breaking the α–β phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).

DNA polymerases catalyze the incorporation of deoxyribonucleoside monophosphate (dNMP) into an existing primer after binding a deoxyribonucleoside triphosphate (dNTP) complementary to a templating base. The catalytic process is thought to begin with deprotonation of the primer 3′-OH by a general base, continue by nucleophilic attack of the α-phosphate of the dNTP leading to a new phosphodiester bond, and finish with release of pyrophosphate after its protonation by a general acid (Fig. 1A) (1). This mechanism has long been established to require two Mg²⁺ ions (A and B) (2). However, visualizing the reaction intermediates using time-resolved X-ray crystallography reveals little movement of the protein but a third and transiently bound Mg²⁺ ion necessary for the DNA synthesis reaction (3, 4). Whether the reaction is driven by the third Mg²⁺ ion or the nucleophile 3′-OH, the identity of a general base responsible for deprotonating the primer 3′-OH remains unclear.Open in a separate windowFig. 1.DNA synthesis reaction. (A) Deprotonation and activation of the primer-end 3′-OH for the nucleophilic attack is hypothesized to be the first step. (B) In the three–Mg²⁺-ion catalysis, the first step appears to break the phosphodiester bond between the α- and β-phosphates of the dNTP. (C) Structural superposition of the reaction-ready (RS) and product state of the WT enzyme in extending the dT primer. The two Mg²⁺ ions (A and B sites) are bound and the 3′-OH is hydrogen-bonded to the transient WatN, which in turn is linked to the bulk solvent via another water molecule. The third Mg²⁺ (C site) is observed only with the products. (D) In the ground state with one Ca²⁺ (green sphere), the 3′-OH is hydrogen-bonded with S113 and not aligned for the inline nucleophilic attack in the absence of the A-site Mg²⁺. The aligned 3′-OH in the RS is shown as semitransparent sticks as a reference. (E) The transient WatN (circled in orange dashes) appears only in the RS and would clash with the C2′ in the GS (wheat) and PS (blue) as indicated by double arrowheads. (F) Sequence alignment of active-site residues around S113 (highlighted in red) of human Pol η, Saccharomyces cerevisiae Pol η (yeast), human Pol ι, human Pol κ, S. cerevisiae Rev1, Escherichia coli Pol IV, and Sulfolobus solfataricus Dpo4. The conserved secondary structures are shown above the sequences.Crystal structures of various DNA polymerases bound to DNA and dNTP, known as ternary complexes, reveal a conserved catalytic center with two Mg²⁺ ions coordinated by the conserved catalytic carboxylates, three phosphates of an incoming dNTP, and the primer 3′-OH (Fig. 1C) (5, 6). DNA polymerase has been likened to a right hand with the palm domain containing the catalytic residues, the finger domain closing on top of the nascent or replicating base pair, and the thumb domain binding the upstream DNA (product) duplex (7, 8). Many DNA polymerases undergo a large conformational change involving closing of the finger domain upon binding a correct dNTP (8), which was proposed to be a rate-limiting step (9). However, the rate of finger domain closing is much faster than the rate of chemical reaction (10–13). Moreover, the finger domain of the Y-family DNA polymerases is already closed even in the absence of an incoming nucleotide (14). Although intradomain motions have been implicated in one example (15), the rate-limiting step is probably the chemical reaction (3, 4, 16, 17).If nucleophilic attack is the first step, deprotonation of the primer 3′-OH by a general base would be essential to initiate the reaction (Fig. 1A). In the more than three decades since the first DNA polymerase structure was determined (7), no residues other than the conserved carboxylates that coordinate the two Mg²⁺ ions have been found to eliminate the catalytic activity when mutated. Computationally, the conserved carboxylates (1, 18) and the incoming nucleotide together with the water molecule bound to the A-site Mg²⁺ (WatA) have each been suggested to deprotonate the 3′-OH (12, 19–21). As the carboxylates and dNTPs are necessary for Mg²⁺ binding and the synthesis reaction, their role in deprotonation is nearly impossible to be experimentally tested.A third Mg²⁺ ion (occupying the C site) has been observed to transiently bind to dNTP in the reactions catalyzed by different DNA polymerases (3, 22–24). Unlike the A- and B-site Mg²⁺ ions, which are coordinated by the catalytic carboxylates, the C-site Mg²⁺ does not contact the enzyme at all. Its low affinity (k_d) matches the minimal Mg²⁺ concentration required for catalysis (3, 4). When two canonical Mg²⁺ ions are bound and reactants are aligned for the inline nucleophilic attack, the third Mg²⁺ ion does not bind readily (3, 4). Only when products are formed is the third Mg²⁺ ion observed to bind between the product DNA and pyrophosphate with four additional water ligands. Yet without the third Mg²⁺, no product can form (4). Its binding is thermal energy (temperature)-dependent, and concurrent with the product formation. The third Mg²⁺ ion may drive dNMP incorporation by breaking the α–β phosphodiester bond of dNTP and pushing the α-phosphate toward the 3′-OH for the new bond formation (4) (Fig. 1B). With the C-site Mg²⁺, deprotonating the 3′-OH is likely favored and does not need a strong general base.The in crystallo analysis of the DNA synthesis reaction catalyzed by human polymerase (Pol) η reveals three reaction states and two potential candidates to deprotonate the nucleophile. Initial dNTP binding with one divalent cation only (B site) leads to the ground state (GS) (Fig. 1D). Binding of the second Mg²⁺ converts the GS to the reactive state (RS), in which the 3′-OH is aligned for the inline nucleophilic attack, and appearance of the third Mg²⁺ (C site) is coupled with the product formation (product state; PS) (3, 4) (Fig. 1 C and E). In the GS, the 3′-OH is within 2.7 Å and hydrogen-bonded to the hydroxyl group of S113 (3). Although S113 is highly conserved among the Y-family polymerases (Fig. 1F) and may facilitate deprotonation of the 3′-OH, removal of the hydroxyl group by mutating S113 to Ala in human Pol η reduces the catalytic efficiency but does not eliminate catalysis (3). In the RS, the 3′-OH is 2.7 Å from a water molecule, which is termed WatN (N for nucleophile) and hypothesized to shuttle the proton off the 3′-OH to the bulk solvent (3). However, preliminary kinetic analysis showed that displacement of the water molecule by the 2′-OH of a ribonucleotide at the primer end leaves catalysis of DNA synthesis unaltered (3).Here we investigate in detail how the 3′-OH is deprotonated in the reaction catalyzed by human Pol η. Combining S113A mutant Pol η (S113A) and modified nucleotides at the primer 3′ end, we measured how the altered environment of the 3′-OH perturbed the steady-state kinetics and reaction process in crystallo. In addition, static structures and dynamics simulation are employed to explain observed kinetic parameters. In contrast to the hypothesis of a specific general base, we find that the proton of the 3′-OH can depart via multiple paths.     

An upper bound for the number of electrons in a large ion

Let E(Z, N) be the ground-state energy of N quantized electrons and a single nucleus of charge Z. For fixed Z, E(Z, N) is independent of N for N ≥ N_critical(Z). Physically, this means that at most N_critical electrons can bind to the nucleus. We prove that N_critical ≤ Z + CZ^a with a = 0.84.