[Substitution effects in phenothiazine radicals. 1. Method for high solvent EPR spectroscopy of phenothiazine radicals].
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Electron Spin Resonance Spectroscopy↗
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Regiospecific and highly stereoselective coupling of 6-(substituted-imidazol-1-yl)purines with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride. Sodium-salt glycosylation in binary solvent mixtures: improved synthesis of cladribine.
Glycosylation of 6-(substituted-imidazol-1-yl)purine sodium salts with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride proceeds with regiospecific formation of the N9 isomers. Base substrates with lipophilic substituents on the C6-linked imidazole moiety are more soluble in organic solvents, and the solubility is further increased with binary solvent mixtures. Selective solvation also diminishes the extent of anomerization of the chlorosugar. Stirred reaction mixtures of the modified-purine sodium salts generated in a polar solvent and cooled solutions of the protected 2-deoxysugar chloride in a nonpolar solvent give 2'-deoxynucleoside derivatives with N9 regiochemistry and enhanced beta/alpha configuration ratios. Application of the binary-solvent methodology with 2-chloro-6-(substituted-imidazol-1-yl)purine salts in cold acetonitrile and the chlorosugar in cold dichloromethane gives essentially quantitative yields of the N9 isomers of beta-anomeric 2'-deoxynucleoside intermediates. Direct ammonolysis (NH(3)/MeOH) of such intermediates or benzylation of the imidazole ring followed by milder ammonolysis of the imidazolium salt gives high yields of the clinical anticancer drug cladribine (2-chloro-2'-deoxyadenosine).
Antineoplastic Agents↗
Torsional rearrangements of aryl-substituted anthracenes measured by phase fluorometry.
We examined the fluorescence phase angle spectra of aryl-substituted anthracenes in viscous solvents. These phase angles reflect the wavelength-dependent lifetimes displayed by the fluorophores. At intermediate viscosities the phase angles increased and decreased in accordance with the valleys and peaks of the structured emission spectra. At lower and higher temperatures the phase angles were independent of emission wavelength, which is equivalent to decay times which are independent of emission wavelength. We attribute the wavelength-dependent phase angles to rotation of the unsaturaled side chains towards a conformation more coplanar with the anthracene ring. By comparative studies with 9,10-di(alpha-naphthylanthracene), 9,10-di-phenylanthracene, and 9-vinylanthracene we showed that lower temperatures are required to inhibit rotation of the smaller phenyl and vinyl substitutents. Since these excited-state rearrangements are sensitive to the motional freedom allowed by the surrounding solvent, we suggest that this phenomenon may serve as a probe of volume fluctuations in macromolecules.
Journal Article↗
Stabilizing and destabilizing effects of placing beta-branched amino acids in protein alpha-helices.
In order to gain greater insight into the effects of beta-branched amino acids on protein alpha-helices, hydrophobic amino acids with varying degrees of beta-branching, including the fully beta-substituted L-2-amino-3,3-dimethylbutanoic acid (ADBA), were incorporated into the protein T4 lysozyme. The unnatural and natural amino acids were substituted at two solvent-exposed alpha-helical sites, Ser 44 and Asn 68, in the protein using the technique of unnatural amino acid mutagenesis. The stabilities of the mutant proteins were determined by using a heat of inactivation assay and from their circular dichroism thermal denaturation curves. Surprisingly, while substitution of the amino acid with the greatest degree of beta-branching, ADBA, destabilizes the protein by 2.5 +/- 0.1 degrees C (0.69 +/- 0.03 kcal/mol) relative to Ala at site 44, the same substitution stabilizes the protein by 1.0 +/- 0.1 degree C (0.27 +/- 0.03 kcal/mol) at site 68. The difference observed at these two positions illustrates the extent to which the local context can mediate the impact of a particular mutation. Molecular dynamics simulations were carried out in parallel to model the structures of the mutant proteins and to examine the energetic consequences of incorporating ADBA. Together, these results suggest that the conformationally restricted beta-branched amino acids are destabilizing, in part, because the beta-branched methyl groups can cause distortions in the local helix backbone. In addition, it is proposed that in some contexts the conformational rigidity of beta-branched amino acids may be stabilizing because it lowers the entropic cost of forming favorable side-chain van der Waals interactions.
Acetonitriles↗
Folding and function of a T4 lysozyme containing 10 consecutive alanines illustrate the redundancy of information in an amino acid sequence.
Single and multiple Xaa----Ala substitutions were constructed in the alpha-helix comprising residues 39-50 in bacteriophage T4 lysozyme. The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable. The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure. The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing. The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1 degrees C relative to wild type at pH 3.0, but reduced by 1.6 degrees C at pH 6.7. In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein. The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding. Also, further evidence is provided that the replacement of fully solvent-exposed residues within alpha-helices with alanines may be a general way to increase protein stability. The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.
Alanine↗
PCM study of the solvent and substituent effects on the conformers, intramolecular hydrogen bonds and bond dissociation enthalpies of 2-substituted phenols.
A PCM continuum model, at the DFT/B3LYP level, is used to study the solvent and substituent effects on the conformers, intramolecular hydrogen bond (HB) enthalpies, (Delta H(intra)s), and O-H bond dissociation enthalpies, (BDEs), in 2-substituted phenols, 2-X-ArOH, in the liquid phase. Two electron-donating (edg) and three electron-withdrawing (ewg) substituents are chosen, involved in a variety of biochemical transformations. Seven solvents, differing in their H-bonding ability and polarity, are selected to model different environmental situations. Very good correlations are found between the computed R(O-H) and nu(O-H) values in solution for all non-HB 2-X-ArOH, showing that the former can be used as an universal molecular descriptor for the latter and vice-versa. In all 2-X-ArOH, the HB parent conformer is the most stable in all media, closely matching frequency experimental data in CCl4. However, for all 2-X-ArO*, the most stable conformer either forms a "reverse"-HB or a HB is not formed, due to the long distance or steric effects. Changes in the stability, in solution, are observed for some of the 2-X-ArO* conformers. The intramolecular HB-strength in solution, Delta H(S,intra), varies significantly with the size of the HB ring formed and the nature of the substituents. Reasonable correlations, derived between the two energetic parameters (BDE(aw,sol) and Delta H(S,intra)) and the solvent ( and a), and/or molecular, [R(O-H) and nu(O-H)] ones, allow for an approximate estimation of the two former from the four latter. 2-X(edg) decrease BDEs (hence, increase the antioxidant efficiency of the solute, too) in all media; 2-X(ewg) present an opposite result. Moreover, an isodesmic reactions study affords total stabilization effect (TSE) values (identical to the Delta[BDE(aw)]s), which are mainly governed by the stabilization of the phenolic radical (SPR) than that of the parent molecule (SPP). Quantitative correlations between the two effects in the TSE in both the gas and the liquid phases are also given. Unlike in the protic solvents, the better stabilization of the radical than the parent species, derived for the 2-X(edg)-ArOH in the aprotic, apolar, and/or low polar solvents, could account well for their smaller BDE(sol)s. An effective antioxidant in solution should involve either one of the two edg in any one of the two latter solvents.
Free Radicals↗
Determination of polychlorinated biphenyls in sediments, using sonication extraction and capillary column gas chromatography-electron capture detection with internal standard calibration.
A sonication technique is presented for the extraction of polychlorinated biphenyls (PCBs) from sediments. In addition, a quantitation scheme is described which allows peak-specific and, in many cases, congener-specific determination of PCBs. PCBs are quantitated by capillary column gas chromatography-electron capture detection, with internal standard calibration. Results utilizing sonication extraction were compared with those obtained by Soxhlet and steam distillation extractions of 3 U.S. Environmental Protection Agency (EPA) quality control sediment samples and 3 lake sediments known to be contaminated with PCBs. Environmental lake sediments were extracted wet, with no drying prior to extraction. Recoveries by each technique varied depending on the sediment sample being extracted and degree of chlorination of PCB congeners. With proper selection of extraction solvent, the sonication technique can recover amounts of PCBs equivalent to and sometimes greater than recoveries by the Soxhlet or steam distillation techniques. A 24-h quiescent period in the extraction solvent between 2 sonications improved extraction efficiency for 2 freeze-dried sediments but did not affect results obtained for 3 environmentally contaminated sediments that were extracted without drying. Replacement of Soxhlet extraction with the sonication technique results in reduced sample preparation time, decreased volumes of solvents and sample, and substitution of common laboratory glassware in place of fragile, expensive Soxhlet glassware. Sonication extraction can also improve precision compared with Soxhlet extraction.
Chromatography, Gas↗
Cytochemical and ultrastructural studies on the synaptonemal complex of rat spermatocytes.
The effects of several dehydration treatments on the synaptonemal complex (SC), histone solubility in 2.0 M NaCl, and histone-DNA interaction in unfixed rat spermatocytes were evaluated. Freeze substitution with ethanol or dehydration with polyethylene glygol resulted in loss of the SC, preservation of histone solubility and DNA-histone salt linkages. Dehydration with ethylene gylcol or hexylene glycol resulted in preservation of SC with a clear delineation of attachment of the chromatin fibrils to the lateral elements, but a loss of histone solubility and histone-DNA linkages. Dehydration to a fifty percent concentration with glycerol with completion of dehydration with ethylene glycol had the same effect but also resulted in an even distribution of chromatin fibrils. Dehydration with glycerol alone resulted in clumping of chromatin and loss of SC structure, histone solubility and histone-DNA linkages. Partial dehydration to a fifty percent concentration with these three solvents followed by freeze substitution with ethanol resulted in the loss of SC structure and histone solubility but the preservation of histone-DNA linkages. It is likely that these nonaqueous solvents affected the histone hydrophobic groups and thereby altered histone conformation and interactions. These alterations, depending on the treatment used, resulted in the loss or preservation of SC, histone solubility and histone-DNA interactions thereby indicating that the hydrophobic interactions of the histones are crucial for the preservation of these feature of meiotic chromosomes. These results also demonstrate that neither does the preservation of the histone-DNA salt linkages suffice for the preservation of the SC nor does their disruption necessarily result in its loss. The lysine-rich histones, particularly that one unique to meiotic cells, may through their interactions play a crucial role in SC structure.
Animals↗
Characterization of endoperoxide and hydroperoxide intermediates in the reaction of pyridoxine with singlet oxygen.
The photosensitized oxidation of vitamin B6, pyridoxine, is investigated by product and kinetic analysis. Singlet oxygen quenching rates, measured by time-resolved laser flash generation of singlet oxygen followed by monitoring singlet oxygen phosphorescence decay, confirm previous observations that pyridoxine is a moderate quencher. The quenching rate for 3-methoxypyridine is 100 times slower than that for 3-hydroxypyridine, indicating the hydroxy moiety is required for efficient quenching. The chemical quenching rate constant, kr, was estimated by comparison with a known singlet oxygen reaction. Results indicate that the chemical quenching rate of pyridoxine dominates the total quenching. The major reaction product in methanol was isolated and characterized by NMR and MS. The data are consistent with a solvent adduct of the substituted 2,5-pyridinedione. At low temperature, two semistable intermediates were characterized by NMR. The data are consistent with a hydroperoxide and endoperoxide. These intermediates suggest initial attack of singlet oxygen para to the hydroxy group followed by either proton transfer to form the hydroperoxide or addition of the peroxide to the imine to form the endoperoxide. In the presence of protic solvents, the solvent adducts to the imine and elimination of water yield the observed 2,5-pyridinedione product.
Hydrogen Peroxide↗
Solvent and concentration effects on the visible spectra of tri-para-dialkylamino-substituted triarylmethane dyes in liquid solutions.
We have characterized the spectroscopy properties of crystal violet (CV+) and ethyl violet (EV+) in liquid solutions as a function of the solvent type and dye concentration. The analysis of how solvent properties and dye concentration affects the electronic spectra of these tri-para-dialkylamino substituted tryarylmethane (TAM+) dyes was performed on the basis of two spectroscopic parameters, namely the difference in wavenumber (deltanu) between the maximum and the shoulder that appears in the short-wavelength side of the respective maximum visible band (deltanu = 1/lambda(shoulder)-1/lambda(max) cm(-1)), and the wavelength of the maximum absorption (lambda(max)). The solvent and the concentration effects on lambda(max) and deltanu have indicated that both solute/solute (ion-pairing and dye aggregation) and solute/solvent (H-bonding type) interactions modulate the shape of the visible electronic spectra of these dyes in solution. In solvent with small dieletric constant (epsilon < approximately 10), the formation of ion-pairs represents a major contribution to the shaping of these spectra. Upon increasing dye concentration the formation of ion-pairs was characterized by an increase in deltanu observed concomitantly with a red shift in lambda(max) In chloroform and chlorobenzene the ion-pair association constant of CV+ and EV+ with Cl- ions were found to be in the order of 10(6) and 10(5) M(-1), respectively. In trichloroethylene the association constant for the CV+Cl- pair was 10(8) M(-1). In water, dye aggregation instead of ion-pairing represents a major contribution to the shaping of the visible spectra of CV+ and EV+. Dye aggregation was indicated by an increase in deltanu observed concomitantly with a blue shift in lambda(max) upon increasing dye concentration. The distinct behavior of deltanu for dye aggregation and ion-pairing as a function of dye concentration can therefore assist in the characterization of these two distinct phenomena. The solute/solvent interactions were studied in a series of polar solvents in which solute/solute interactions do not occur in any detectable extent. The dependence found for deltanu as a function of the Kamlet-Tafts solvatochromic parameters (alpha, beta and pi*) is in keeping with previous inferences indicating that the splitting in the overlapped absorption band of CV+ and EV+ in hydroxilated solvents arises from a perturbation in the molecular symmetry induced by hydrogen bonding (donor-acceptor) type interactions with solvent molecules. A distinction between the effects of solute/solute and solute/solvent interactions on the visible spectra of these dyes is provided.
Gentian Violet↗
Solvent and solvent proton dependent steps in the galactose oxidase reaction.
Solvent and solvent proton dependent steps involved in the mechanism of the enzyme galactose oxidase have been examined. The deuterium kinetic solvent isotope effect (KSIE) on the velocity of the galactose oxidase catalyzed oxidation of methyl beta-galactopyranoside by O2 was measured. Examination of the thermodynamic activation parameters for the reaction indicated that the isotope effect was attributable to a slightly less favorable delta H value, consistent with a KSIE on proton transfer. A detailed kinetic analysis was performed, examining the effect of D2O on the rate of reaction over the pH range 4.8-8.0. Both pL-rate profiles exhibited bell-shaped curves. Substitution of D2O as solvent shifted the pKes values for the enzymic central complex: pKes1 from 6.30 to 6.80 and pKes2 from 7.16 to 7.35. Analysis of the observed shifts in dissociation constants was performed with regard to potential hydrogenic sites. pKes1 can be attributed to a histidine imidazole, while pKes2 is tentatively assigned to a Cu2+-bound water molecule. A proton inventory was performed (KSIE = +1.55); the plot of kcat vs. mole fraction D2O was linear, indicating the existence of a single solvent-derived proton involved in a galactose oxidase rate-determining step (or steps). The pH dependence of CN- inhibition was also examined. The Ki-pH profile indicated that a group ionization, with pKa = 7.17, modulated CN- inhibition; Ki was at a minimum when this group was in the protonated state. The inhibition profile followed the alkaline limit of the pH-rate profile for the enzymic reaction, suggesting that the group displaced by CN- was also deprotonating above pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)
Cyanides↗
The AD1 transactivation domain of E2A contains a highly conserved helix which is required for its activity in both Saccharomyces cerevisiae and mammalian cells.
A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.
Amino Acid Sequence↗
Coordination geometries for monovalent and divalent metal ions in [His121]azurin--studies using perturbed angular correlations of gamma-rays from 111Ag and 111mCd.
The structural details of the metal site in the [His121]azurin mutant from Alcaligenes denitrificans where the axial methionine has been replaced by a histidine have been studied after substitution with the divalent cadmium ion and the monovalent silver ion. The studies have been carried out in solution using the technique of perturbed angular correlations of gamma-rays (PAC) of the two isotopes, 111Ag and 111mCd. In the pH range 6-9, the PAC spectra for cadmium-substituted [His121]azurin reveals a pH-independent equilibrium between two different metal-coordination geometries. Interpretation of the PAC data shows agreement between the dominating coordination geometry and that derived from X-ray diffraction on the Cu(II)[His121] azurin at high pH (Messerschmidt, A., unpublished results). Thus, it appears likely that cadmium for this geometry is four coordinated to the ligands His46, His117, Cys112, and His121. The other geometry is best interpreted as a substitution of His121 by a solvent water ligand. These interpretations stem from predictions of the experimentally determined nuclear quadrupole interactions (NQI) via the simple angular overlap model (AOM). At low pH (3.8), the concentration of the former species is reduced to 50% of its high pH value suggesting a pK of about 4 for His121. Two different coordination geometries have also been observed for the Cu(II) protein and assigned a type 1.5 and a type 1 copper site [Kroes, S. J., Hoitink, C. W. G., Andrew, C. R., Ai, J., Sanders-Loehr, J., Messerschmidt, A., Hagen, W. R. & Canters, G. W. (1996a) Eur. J. Biochem. 240, 342-351]. For silver-substituted [His121]azurin, several notable changes occur relative to the cadmium-substituted protein. At least four different metal-coordination geometries exist for silver[His121]azurin in the pH range 4-8. Changes in the population of these coordination sites occurs between pH 4 and pH 5, and pH 5 and pH 6. Furthermore, in contrast to the cadmium-substituted protein, only a single coordination geometry is present above pH 6. The change in population occurring between pH 5 and pH 6 suggests ionization of a non-coordinating histidine, here proposed as His121. The change in population at low pH could then be due to protonation of an additional coordinating histidine such as His46 or His117. The single coordination geometry existing at pH values above 6 for the silver protein cannot within our model calculations be described with His121 coordinated. However, it can be described with a coordinated water molecule but in a different angular position than for His121 in the copper protein (Messerschmidt, A., unpublished results). The reduced tendency for silver to coordinate His121 is in agreement with the general trend of lower pK values for ligands coordinating to monovalent ions relative to divalent ions. In conclusion, this work demonstrates that mutation of Met121 to other amino acid residues opens the possibility of other coordination geometries than the rigid three-coordinated structure observed for wild-type azurin, especially the possibility of increasing the coordination number by either solvent water ligands or the substituting amino acid. Furthermore, it opens up the possibility for different coordination geometries for monovalent and divalent metal ions as observed here and previously for the [Leu121]azurin mutant [Bauer, R., Danielsen, E., Hemmingsen, L., Bjerrum, M. J., Hansson, O. & Singh, K. (1997) J. Am. Chem. Soc. 119, 157-163].
Azurin↗
6-bromopurine nucleosides as reagents for nucleoside analogue synthesis.
Surprisingly facile direct substitution reactions with acetyl-protected 6-bromopurine nucleosides are described. Included in the series of bromonucleosides studied is the guanosine derivative N(2)-2',3',5'-tetraacetyl-6-bromopurine ribonucleoside, the synthesis of which is reported here for the first time. Brominated nucleosides had not previously been considered optimal substrates for S(N)Ar reactions given the general reactivity trend for halogenated aromatic systems (i.e. F > Cl > Br > I). However, even weakly nucleophilic aromatic amines give high yields of the substitution products in polar solvents with these 6-bromopurine nucleosides. For primary aromatic amines, secondary aliphatic amines, and imidazole, reaction takes place only at C6, with no effect on the acetyl-protected ribose. In addition, we report the first synthesis of 3',5'-di-O-acetyl-6-bromopurine-2'-deoxyribonucleoside and its reaction with an arylamine in MeOH in the absence of added metal catalyst. Thus, C6-arylamine derivatives of both adenosine and 2'-deoxyadenosine can be prepared via simple S(N)Ar reactions with the corresponding 6-bromo precursor. We also describe high yielding and C6-selective substitution reactions with 6-bromonucleosides using alcohol and thiol nucleophiles in the presence of added base (DBU). Finally, C6-bromonucleosides are shown to be readily hydrogenated to give purine or 2-aminopurine products in good yield. This work increases the arsenal of reactions and strategies available for the synthesis of nucleoside analogues as potential biochemical tools or new therapeutics.
Amines↗
DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo[4,5- f]quinoline at the C-8 and N2 atoms of guanine.
DNA adduct formation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been investigated by 32P-postlabeling. Similar adduct profiles were observed from calf thymus DNA modified in vitro with the putative carcinogenic metabolite N2-acetoxyamino-3-methylimidazo[4,5-f]-quinoline (N-acetoxy-IQ) and from hepatic DNA of rats treated with IQ. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) accounted for approximately 90% of the total adducts observed in calf thymus DNA under postlabeling conditions where ATP was limiting; however, 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5- f]quinoline (dG-N2-IQ) was detected only when DNA was labeled with excess ATP. Under these labeling conditions, dG-C8-IQ and dG-N2-IQ accounted for approximately 75% and 7% of the total adducts, respectively. Five other spots accounted for the remaining radioactivity. Comparable results were obtained from rat liver DNA. Following DNA adduct enrichment by solid phase extraction, dG-C8-IQ and dG-N2-IQ accounted for 60-76% and 10-13%, respectively, of the total adducts in rat liver. The adduct profiles obtained from reaction of 2'-deoxyguanosine 3'-monophosphate (dG-3'-PO4-) with the photoactivated azide derivative of IQ, 2-azido-3-methylimidazo[4,5-f]quinoline (N3-IQ), were qualitatively similar to those obtained by reaction with N-acetoxy-IQ. The C-8 and N2 adducts were the only reaction products detected. The reactivity and sites of adduct substitution were dependent upon solvent conditions and pH, with increasing adduct formation under alkaline pH. The chemical reactivity of photoactivated N3-IQ with dG-3'-PO4- was significantly greater than that of N-acetoxy-IQ when reactions were conducted in water, in citrate buffer (pH 5.0), or in phosphate buffer (pH 7.4). Increased reactivity was attributed to increased levels of dG-C8-IQ adduct formation, except for reactions conducted in citrate buffer (pH 5.0), where there was a proportional increase in both C-8 and N2 guanine adducts. However, the chemical reactivity of these two IQ derivatives and their sites of dG substitution were identical when the reactions were conducted in phosphate buffer (pH 9.0). The ratio of the dG-N2-IQ adduct to the total adducts increased at alkaline pH in reactions involving N3-IQ, but the ratio was not affected by a change in the pH of the medium for reactions with N-acetoxy-IQ. The ratio of the dG-N2-IQ adduct to the total adducts also increased as a function of phosphate concentration for reactions involving both N-acetoxy-IQ and N3-IQ.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Role of water activity on the rates of acetyl phosphate and ATP hydrolysis.
The rates of hydrolysis of acetyl phosphate in the presence of 0.1 M NaOH and of ATP in the presence of either 1 M HCl or 1 M NaOH were measured at different temperatures and in the presence of different concentrations of the organic solvents dimethyl sulfoxide or ethylene glycol. Under all conditions tested, there was a progressive increase in the rate constant of hydrolysis of both phosphate compounds as the water activity of the medium was decreased by the addition of organic solvents. At 25 degrees C, substitution of 70% of the water of the medium by dimethyl sulfoxide promoted an increase of two orders of magnitude in the rate constant of acetyl phosphate hydrolysis. In the presence of 80% and 90% dimethyl sulfoxide the rate of acetyl phosphate hydrolysis increased by more than two orders of magnitude and was so fast that it could not be measured with the method used. The effect of organic solvents on the rate of ATP hydrolysis was less pronounced than that observed for acetyl phosphate hydrolysis. At 30 degrees C, substitution of 90% of water by an organic solvent promoted a 4-6-fold increase of the rate of ATP hydrolysis. Acceleration of either acetyl phosphate or ATP hydrolysis rates was promoted by a decrease in both activation energies (Ea) and in entropies of activation delta S. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the Ca2+-ATPase of sarcoplasmic reticulum and the F1-ATPase of mitochondria.
Adenosine Triphosphate↗
Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.
The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.
Actinomycetales↗
Natriuretic peptide receptor A activation stabilizes a membrane-distal dimer interface.
We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.
Atrial Natriuretic Factor↗