Search

Dibromoethane is a widespread, persistent organic pollutant. Biochars are known mediators of reductive dehalogenation by layered Fe II -Fe III hydroxides (green rust), which can reduce 1,2-dibromoethane to innocuous bromide and ethylene. However, the critical characteristics that determine mediator functionality are lesser known. Fifteen biochar substrates were pyrolyzed at 600 °C and 800 °C, characterized by elemental analysis, X-ray photo spectrometry C and N surface speciation, X-ray powder diffraction, specific surface area analysis, and tested for mediation of reductive debromination of 1,2-dibromoethane by a green rust reductant under anoxic conditions. A statistical analysis was performed to determine the biochar properties, critical for debromination kinetics and total debromination extent. It was shown that selected plant based biochars can mediate debromination of 1,2-dibromoethane, that the highest first order rate constant was 0.082/hr, and the highest debromination extent was 27% in reactivity experiments with 0.1 µmol (20 µmol/L) 1,2-dibromoethane, ≈ 22 mmol/L Fe II GR , and 0.12 g/L soybean meal biochar (7 days). Contents of Ni, Zn, N, and P, and the relative contribution of quinone surface functional groups were significantly (p < 0.05) positively correlated with 1,2-dibromoethane debromination, while adsorption, specific surface area, and the relative contribution of pyridinic N oxide surface groups were significantly negatively correlated with debromination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The use of alkynyl gold(I) complexes covers different research fields, such as bioinorganic chemistry, catalysis, and material science, considering the luminescent properties of the complexes. Regarding this last application, we report here the synthesis of three novel dinuclear gold(I) complexes of the general formula [(diNHC)(Au-C≡CPh) 2 ]: two Au-C≡CPh units are connected by a bridging di(N-heterocyclic carbene) ligand, which should favor the establishment of semi-supported aurophilic interactions. The complexes can be easily synthesized through mechanochemistry upon reacting the pristine dibromido complexes [(diNHC)(AuBr) 2 ] with phenylacetylene and KOH. Interestingly, we were also able to isolate the monosubstituted complex [(diNHC)(Au-C≡CPh)(AuBr)]. The gold(I) species were fully characterized by multinuclear NMR spectroscopy and mass spectrometry. The emission properties were also evaluated, and the salient data are comparable to those of analogous compounds reported in the literature.

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (k cat /K m ) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Interactions between enzymes and small molecules lie in the center of many fundamental biochemical processes. Their analysis using molecular dynamics simulations have high computational demands, geometric approaches fail to consider chemical forces, and molecular docking offers only static information. Recently, we proposed to combine molecular docking and geometric approaches in an application called CaverDock. CaverDock is discretizing enzyme tunnel into discs, iteratively docking with restraints into one disc after another and searching for a trajectory of the ligand passing through the tunnel. Here, we focus on the practical side of its usage describing the whole method: from getting the application, and processing the data through a workflow, to interpreting the results. Moreover, we shared the best practices, recommended how to solve the most common issues, and demonstrated its application on three use cases.

Conformational isomerism plays a crucial role in defining the physical and chemical properties and biological activity of molecules ranging from simple organic compounds to complex biopolymers. However, it is often a significant challenge to differentiate and separate these isomers experimentally as they can easily interconvert due to their low rotational energy barrier. Here, we use the momentum correlation of fragment ions produced after inner-shell photoionization to distinguish conformational isomers of 1,2-dibromoethane (C 2 H 4 Br 2 ). We demonstrate that the three-body breakup channel, C 2 H 4 + + Br + + Br + , contains signatures of both sequential and concerted breakup, which are decoupled to distinguish the geometries of two conformational isomers and to quantify their relative abundance. The sensitivity of our method to quantify these yields is established by measuring the relative abundance change with sample temperature, which agrees well with calculations. Our study paves the way for using Coulomb explosion imaging to track subtle molecular structural changes.

In this work, a one-pot, telescopic approach is described for the combinatorial library of thiazolidine-2-imines. The synthetic manipulation proceeds smoothly via the reaction of 2-aminopyridine/pyrazine/pyrimidine with substituted isothiocyanates followed by base catalyzed ring closure with 1,2-dibromoethane to obtain thiazolidine-2-imines with broad substrate scope and high functional group tolerance. The synthetic strategy merges well with the thiourea formation followed by base catalyzed ring closure reaction for the thiazolidine-2-imine synthesis in a more modular and straightforward approach. The synthetic procedure reported herein represents a cleaner route toward thiazolidine-2-imines as compared to traditional methodologies. Moreover, the biological significance of combinatorially synthesized thiazolidin-2-imines has been investigated for their use as possible inhibitors for acetyl cholinesterase through molecular docking studies.

We performed a model-based investigation to simultaneously predict the evolution of concentration, as well as stable carbon and bromine isotope fractionation during 1,2-dibromoethane (EDB, ethylene dibromide) transformation in a closed system. The modelling approach considers bond-cleavage mechanisms during different reactions and allows evaluating dual carbon-bromine isotopic signals for chemical and biotic reactions, including aerobic and anaerobic biological transformation, dibromoelimination by Zn(0) and alkaline hydrolysis. The proposed model allowed us to accurately simulate the evolution of concentrations and isotope data observed in a previous laboratory study and to successfully identify different reaction pathways. Furthermore, we illustrated the model capabilities in degradation scenarios involving complex reaction systems. Specifically, we examined (i) the case of sequential multistep transformation of EDB and the isotopic evolution of the parent compound, the intermediate and the reaction product and (ii) the case of parallel competing abiotic pathways of EDB transformation in alkaline solution.

A four-component reaction of aryldiazonium tetrafluoroborates, sulfur dioxide, 1,2-dibromoethane, and hydrazines under metal-free conditions is described, providing a novel and efficient approach to 2-arylsulfonyl hydrazones. This transformation proceeds smoothly via insertion of sulfur dioxide under mild conditions with good functional group tolerance.

We report an experimental and computational study of the photodecomposition pathways of a prototypical gem-dihalide, 1,1-dibromoethane (1,1-EDB), in the condensed phase. Following photolysis of the matrix isolated parent compound in Ar at 5 K, photoproducts are observed corresponding to Br2 elimination (+ C2H4 or C2H2) and HBr elimination (+ vinyl bromide). The elimination products are observed in the matrix as complexes. In contrast to our recent studies of the photolysis of matrix isolated polyhalomethanes, no evidence for the iso-1,1-EDB species is found, although studies of the matrix isolated 1,1-dibromo-2,2,2-trifluoroethane analogue show that the isomer is the dominant photoproduct. These results are examined in the light of theoretical studies that have characterized in detail the 1,1-EDB potential energy surface (PES). For Br2 elimination, a pathway from the isomer on the singlet PES is found which involves a simultaneous Br2 loss with 1,2-hydrogen shift; this pathway lies lower in energy than a concerted three-center elimination from the parent 1,1-EDB. For HBr elimination, our previous theoretical studies [Kalume, A.; George, L.; Cunningham, N.; Reid, S. A. Chem. Phys. Lett. 2013, 556, 35-38] have demonstrated the existence of concerted (single-step) and sequential pathways that involve coupled proton and electron transfer, with the sequential pathway involving the isomer as an intermediate. Here, more extensive computational results argue against a simple radical abstraction pathway for this process, and we compare experimental and computational results to prior results from the photolysis of the structural isomer, 1,2-EDB. These steady-state experiments set the stage for ultrafast studies of the dynamics of this system, which will be important in unraveling the complex photodecomposition pathways operative in condensed phases.

Several studies with bacteria and in vitro mammalian systems have provided evidence of the roles of two thiol-based conjugation systems, glutathione (GSH) transferase and O(6)-alkylguanine DNA-alkyltransferase (AGT), in the bioactivation of the bis-electrophiles 1,2-dibromoethane and 1,2,3,4-diepoxybutane (DEB), the latter an oxidation product of 1,3-butadiene. The in vivo relevance of these conjugation reactions to biological activity in mammals has not been addressed, particularly with DEB. In this work, we used transgenic Big Blue mice, utilizing the cII gene, to examine the effects of manipulation of conjugation pathways on liver mutations arising from dibromoethane and DEB in vivo. Treatment of the mice with butathionine sulfoxime (BSO) prior to dibromoethane lowered hepatic GSH levels, dibromoethane-GSH DNA adduct levels (N(7)-guanyl), and the cII mutation frequency. Administration of O(6)-benzylguanine (O(6)-BzGua), an inhibitor of AGT, did not change the mutation frequency. Depletion of GSH (BSO) and AGT (O(6)-BzGua) lowered the mutation frequency induced by DEB, and BSO lowered the levels of GSH-DEB N(7)-guanyl and N(6)-adenyl DNA adducts. Our results provide evidence that the GSH conjugation pathway is a major in vivo factor in dibromoethane genotoxicity; both GSH conjugation and AGT conjugation are major factors in the genotoxicity of DEB. The latter findings are considered to be relevant to the carcinogenicity of 1,3-butadiene.

The sensitivity of the 1s X-ray emission and high-energy-resolution fluorescence-detected X-ray absorption spectroscopies (XES and HERFD-XAS) to resolve the variations in the chemical state (electronic structure and local coordination) of Br has been investigated for a selected set of compounds including NaBrO3, NH4Br and C2H4Br2 (1,2-dibromoethane). For the Br K-edge XAS, employing the HERFD mode significantly increases the energy resolution, which demonstrates that with a crystal spectrometer used as a detector the absorption technique becomes a more powerful analytical tool. In the case of XES, the experimental results as well as the density functional theory (DFT) modeling both show that the chemical sensitivity of the main 1s diagram emission lines (Kα1,2 and Kβ1,3) is rather limited. However, the valence-to-core (Kβ2) region of XES displays significant shape and intensity variations, as expected for transitions having the same final states as those of photoemission spectroscopy. The spectra are in good agreement with the molecular orbital description delivered by DFT calculations. Calculations for an extended series of Br compounds confirm that valence-to-core XES can serve as a probe for chemical analysis, and, being a hard X-ray photon-in/photon-out technique, it is particularly well-suited for in situ investigations of molecular transformations, even on the ultrafast time scales down to femtosecond time resolution.

Haloalkane dehalogenases are microbial enzymes that convert a broad range of halogenated aliphatic compounds to their corresponding alcohols by the hydrolytic mechanism. These enzymes play an important role in the biodegradation of various environmental pollutants. Haloalkane dehalogenase LinB isolated from a soil bacterium Sphingobium japonicum UT26 has a relatively broad substrate specificity and can be applied in bioremediation and biosensing of environmental pollutants. The LinB variants presented here, LinB32 and LinB70, were constructed with the goal of studying the effect of mutations on enzyme functionality. In the case of LinB32 (L117W), the introduced mutation leads to blocking of the main tunnel connecting the deeply buried active site with the surrounding solvent. The other variant, LinB70 (L44I, H107Q), has the second halide-binding site in a position analogous to that in the related haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94. Both LinB variants were successfully crystallized and full data sets were collected for native enzymes as well as their complexes with the substrates 1,2-dibromoethane (LinB32) and 1-bromobutane (LinB70) to resolutions ranging from 1.6 to 2.8 Å. The two mutants crystallize differently from each other, which suggests that the mutations, although deep inside the molecule, can still affect the protein crystallizability.

EDB (1,2-dibromoethane) is frequently detected at sites impacted by leaded gasoline. In reducing environments, EDB is highly susceptible to abiotic degradation. A study was conducted to evaluate the potential of compound-specific isotope analysis (CSIA) in assessing abiotic degradation of EDB in sulfate-reducing environments. Water containing EDB was incubated in sealed vials with various combinations of Na(2)S (<0.7 mM) and mackinawite (FeS) (180 mM). Degradation rates in vials containing FeS exceeded those in Na(2)S-only controls. In the presence of FeS, first-order constants ranged from 0.034 ± 0.002 d(-1) at pH 6 to 0.081 ± 0.005 d(-1) at pH 8.5. In the presence of FeS, products from reductive debromination (ethylene) and from S(N)2 substitution with S(II) nucleophiles were detected (1,2-dithioethane, DTA). Relatively high yields of DTA suggested that the S(N)2 reactions were not mediated by HS(-) only but likely also included reactions mediated by FeS surface. Significant carbon isotope effects were observed for nucleophilic substitution by HS(-) (ε = -31.6 ± 3.7‰) and for a combination of reductive and substitution pathways in the presence of FeS (-30.9 ± 0.7‰), indicating good site assessment potential of CSIA. The isotope effects (KIEs) observed in the presence of FeS corroborated the predominance of S(N)2 substitution by nucleophiles combined with two-electron transfer reductive debromination.

Adsorbates on metals, but not previously on semiconductors, have been observed to display long-range repulsive interactions. On metals, due to efficient dissipation, the repulsions are weak, typically on the order of 5 meV at 10 Å. On the 7×7 reconstruction of the Si(111) surface, charge transport through the surface has been demonstrated by others using charge injection by STM tips. Here we show that for both physisorbed brominated molecules, and for chemisorbed Br-atoms, induced charge-transfer in the Si(111)-7×7 surface can lead to a strong repulsive interaction between adsorbates, calculated as 200 meV at 13.4 Å. This large repulsive interaction must be channeled through the surface since it causes widely spaced "one-per-corner-hole" patterns of physisorption (three cases--directly observed here) and subsequent chemisorption (four cases observed). The patterns were observed by ultrahigh vacuum scanning tunneling microscopy for four different brominated hydrocarbon adsorbates; 1,2-dibromoethane, 1-bromopropane, 1-bromopentane, and bromobenzene, deposited individually on the surface. In every case, adsorbates were overwhelmingly more likely to be found singly than multiply adjacent to a corner-hole, constituting a distinctive pattern having a probability p = 7 × 10(-5) compared to a random distribution.

Objective: To design and synthesize novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonists., Methods: A series of novel PPARgamma agonists were designed based on the binding character of PPARgamma agonists and the distribution of pharmacophore. The target compounds were synthesized using p-hydroxybenzaldehyde, 1, 2-dibromoethane, phenol, hydantoin and 2-thiohydantoin as materials., Results: Twelve compounds were synthesized by etherification and Knoevenagle condensation., Conclusion: The target compounds were efficiently synthesized under mild condition and the structures of the target compounds were confirmed by 1HNMR and MS.

The quaterisation process of 1,2-dibromoethane and pyridine is in situ traced by electronic absorption spectrum. Two absorption peaks, induced by mono- and bis-pyridinium salt of 1,2-dibromoethane, appear at 429 nm and 313 nm, respectively. To explain the phenomena, several kinds of alkyl bromides with special structures were selected and compared by experimental measurement and theoretical calculation. The results indicate that for mono-pyridinium salt of 1,2-dibromoethane, the electron donor property of ortho-bromine group increases the electron cloud density of the carbon atom associated with pyridinium cation, which induces red-shift of absorption wavelength.

Porous Vycor is examined by measuring CH(2)Br(2) adsorption in situ with small-angle X-ray scattering. When a class of pores fills with condensed vapors of this particular adsorbate it ceases to act as scatterer and only the remaining empty pores produce a measurable intensity. By determining a number of scattering curves at various relative pressure loadings details on the structure of the glass as well as on the adsorption/desorption mechanism are obtained. Pore chord length and specific surface area are estimated from Porod tangent analysis to 78 A and 108 m(2)/g. Comparison of the results with those reported for N(2) and Ar adsorption is also given. The role of network effects on the shape of the hysteresis loop is considered and the pore-blocking hypothesis is verified from the scattering spectra. The pore connectivity is calculated to 5.6. The X-ray data are further treated with the inverse Fourier transformation technique. Pore-size distributions are extracted and weighed against the prediction of the Kelvin equation.

The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and are known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O6 -alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein cross-link formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate because of the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH cross-link formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein cross-links and enhanced mutagenesis.

The molecular structure and conformational properties of 1,2-dibromoethyl-trichlorosilane (CH2BrCHBrSiCl3) have been investigated using gas-phase electron diffraction (GED) data recorded at a temperature of 100 degrees C, together with ab initio molecular orbital (MO) and density functional theory (DFT) calculations, infrared (IR) and Raman spectroscopy in the liquid and solid phases, and normal coordinate analysis (NCA). The molecule exists in the gas- and liquid phases as a mixture of three conformers, gauche(-) [G(-)], with a refined torsion angle phi(BrCCBr)=-71(6) degrees, anti [A], with a torsion angle phi(BrCCBr) approximately -170 degrees , and gauche(+) [G(+)], with a torsion angle phi(BrCCBr) approximately +70 degrees . The second torsion angle of importance, the rotation about the CSi bond, has been refined to a value of +175(13) degrees . Torsion angles were only refined for the more abundant G(-) conformer. In the solid phase, only the G(-) conformer was observed. The temperature-dependent Raman spectra have provided an estimate of the relative conformational entropies, DeltaS. The obtained composition from GED refinements was (%) G(-)/A/G(+)=64(27)/23(13)/13(18) (values with estimated 2sigma uncertainties), giving a conformational stability order in agreement with both the Raman enthalpy measurements and the ab initio MO and DFT calculations using the 6-311G(d) basis set and scaled zero-point energies. Relevant structural parameter values obtained from the GED refinements (with the ab initio HF values used as constraints) were as follows (G(-) values with estimated 2sigma uncertainties): bond lengths (r(g)):r(C-C)=1.501(18)A, r(SiC)=1.865(15)A, r(CBr)=1.965(8)A (average), r(SiCl)=2.028(3)A (average). Bond angles ( anglealpha):angleCCSi=114.1(33) degrees , angleC1C2Br=114.0(21) degrees , angleCSiCl=109.6(7) degrees (average). Experimental IR/Raman and obtained vibrational wavenumbers based on both the unscaled, fixed-scaled as well as the scale-refined quantum-mechanical force fields [HF/6-311G(d)] are presented. The results are discussed and compared with some similar molecules from the literature.

Photolyses of matrices of either BrCHCHBr/NO2/Ar or ClCHCHCl/NO2/Ar using quartz-filtered radiation (lambda>240 nm) led to the appearance of infrared bands attributable to carbonyl, carbon monoxide, and ketene species; no bands belonging to a precursor complex NO2cdots, three dots, centeredXCHCHX (where X=Br or Cl) were observed upon matrix deposition. The possible reaction pathway is discussed.

Mechanisms of toxicity continue to be important in developing rational strategies to deal with chemicals present in the environment. Understanding and predicting toxicity have also become a critical step in the process of drug development. Covalent binding of chemicals to macromolecules is one aspect of toxicity, and the principles and outcomes of the process are considered. Two examples of chemicals for which several aspects of metabolism and reactions are understood are aflatoxin B(1) and polyhalogenated olefins. Ethylene dibromide is a compound that is activated to genotoxic half-mustards by conjugation with glutathione or the DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT). The AGT reaction is unusual, in that crosslinking of the protein to DNA increases mutagenicity. One of the involved mechanisms is formation of N(7)-guanyl crosslinks and depurination to produce G-->T transversions; other reactions appear to yield the additional mutagenic events. The phenomenon of thiol conjugation to increase mutagenicity is widespread among bis-electrophiles.

[reaction: see text] Partition coefficient measurements and experiments with a dye show that a new fluorous "phase vanishing reaction" described by Jana and Verkade occurs by an extractive mechanism. This mechanism is contrasted with the original diffusive phase-vanishing reactions introduced by Ryu and co-workers.

It has been proposed that the DNA repair protein O6-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly136-Arg147 peptide cross-linked to a Gua at the N7 position, with the site of reaction being the active site Cys145 as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N7 adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N7 atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N7-alkylation by alkyltransferase-S-CH2CH2Br and depurination, plus another as yet uncharacterized system(s).

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state k(cat) with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Cl alpha) from 1,2-dichloroethane interacts with Trp125 and the Cl beta interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in k(cat) could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The k(cat) for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Cl alpha or Br alpha) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

The 2-chloroethyl methanesulfonate (2ClEMS)-induced alcohol dehydrogenase (Adh) null germline mutation frequency in treated Drosophila melanogaster second instar larval gonia was two orders of magnitude greater than the spontaneous mutation frequency. DNA sequence analysis of 83 Adh null mutations showed that 40 mutations of independent origin were at 23 sites in the Adh gene. The mutation spectrum contained only GC-->AT transitions with 35 mutations (87.5%) at the middle or 3' guanine. In addition, characteristics of glutathione (GSH)-mediated bioactivation were determined for 2ClEMS in vitro. Rates of GSH-mediated conjugation, catalyzed by purified rat liver glutathione-S-transferase (GST), and binding of [35S]GSH-mediated conjugation products to calf thymus DNA were determined for 2ClEMS, 1,2-dichloroethane (EDC) and 1,2-dibromoethane (EDB). The relative rates of GSH-mediated conjugation were the following: 5 mM EDB > 40 mM 2ClEMS > 40 mM EDC. A similar trend was observed for DNA binding of the [35S]GSH-mediated conjugation products when differences in mutagen concentration were considered: EDB > 2ClEMS > EDC. The ratios of DNA binding to GSH conjugation calculated for EDB, EDC and 2ClEMS were 6.8 x 10(-5), 9.3 x 10(-5) and 19.1 x 10(-5), respectively. A narrow range, less than a 3-fold difference, in the ratios of DNA binding to GSH conjugation indicates that the bioactivation of 2ClEMS is mediated by the same mechanism as EDB and EDC. Consequently, 2ClEMS, EDC and EDB may induce a specific mutation in premeiotic germ cells.

The oligomer d(TGCTGCAAG) corresponds to a region of bacteriophage M13mp18 DNA where mutations have been found to be induced by S-(2-chloroethyl)glutathione (glutathione, GSH) [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., & Guengerich, F. P. (1991) J. Biol. Chem. 267, 6672-6679]. This oligomer was prepared with the central G replaced by S-(2-N7-guanylethyl)-GSH or N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester; these derivatives were purified by HPLC and by affinity chromatography in the latter case. UV mixing and CD spectroscopy studies showed no evidence for preferred pairing of the S-(2-N7-guanylethyl)GSH moiety to any base other than C. UV melting studies of duplexes were performed with complementary strands containing the normal C, as well as the three mismatches (T, A, and G), across from the adducted base. Thermal stabilities were reduced in all cases when G was replaced by either N7-guanyl adduct; the C-containing complement was still the most stable. The reduced stability of the duplex d(TGCTG*CAAG)/d(CTTGCAGCA), where S-(2-N7-guanylethyl)-GSH corresponds to G*, was characterized by an increase in delta G zero of 1.4-2.0 kcal mol-1 (in the range of 25-37 degrees C) relative to the unadducted duplex. van't Hoff analysis of concentration-dependent melting experiments indicated that the delta H zero of the duplex was actually more favorable when this adduct was introduced (delta delta H zero = 13 kcal mol-1), but the decreased thermal stability was due to the entropic component. Similar results were observed when G* was N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester. Under the conditions used, the overall relative stabilities of the oligomeric duplexes containing various base pairs do not indicate that S-(2-N7-guanylethyl)GSH would contribute to a higher frequency of T misinsertion than G. The possibility that ionization at the guanine N1 position may be involved in mutagenesis by N7-guanyl adducts is considered.