Structural properties of the neutral and monoanionic forms of xanthosine,highly relevant to their substrate properties with various enzyme systems

The monoanions of the 6-oxopurines guanine (Gua) and hypoxanthine (Hx), and their nucleosides, pKa approximately 9 due to dissociation of the N(1)-H, are predominantly in their neutral forms at physiological pH. By contrast, the monoanions of the 6-oxopurine xanthine (Xan) and xanthosine (Xao), were long ago proposed to involve dissociation of the N(3)-H, with pKa values of 7.5 and 5.7, respectively, so that, at physiological pH, the former is mixture of the neutral and monoanionic species, and the latter predominantly the monoanion. We have employed multi-dimensional heteronuclear NMR spectroscopy, which fully confirms the proposed mode of monoanion formation in Xao (and, by implication, in Xan), further supported by the results of ab initio quantum mechanical calculations, and additionally extended to determination of the preferred conformational parameters in solution for the neutral and monoanionic species. These findings are highly relevant to the modes of binding, and to the substrate properties, of Xan, Xao and its 5'-phosphate (XMP) in numerous enzyme systems, hitherto virtually ignored, and illustrated by several concrete examples.     

Variations of the tautomeric preferences and π-electron delocalization for the neutral and redox forms of purine when proceeding from the gas phase (DFT) to water (PCM)

Quantum-chemical calculations were performed for all possible nine neutral tautomers of purine and their oxidized and reduced forms in water {PCM//DFT(B3LYP)/6?311+G(d,p)} and compared to those in the gas phase {DFT(B3LYP)/6?311+G(d,p)}. PCM hydration influences geometries, π-electron delocalization, and relative energies of purine tautomers in different ways. Generally, the harmonic oscillator model of electron delocalization (HOMED) indices increase when proceeding from the gas phase to aequeous solution for the neutral and redox forms of purine. Their changes for the neutral and oxidized tautomers are almost parallel to the relative energies showing that aromaticity plays an important role in the tautomeric preferences. Tautomeric stabilities and tautomeric preferences vary when proceeding from the gas phase to water indicating additionally that intra- and intermolecular interactions affect tautomeric equilibria. The tautomeric mixture of neutral purine in the gas phase consists mainly of the N9H tautomer, whereas two tautomers (N9H and N7H) dominate in water. For oxidized purine, N9H is favored in the gas phase, whereas N1H in water. A gain of one electron dramatically changes the relative stabilities of the CH and NH tautomers that C6H and C8H dominate in the tautomeric mixture in the gas phase, whereas N3H in water. These variations show exceptional sensitivity of the tautomeric purine system on environment in the electron-transfer reactions.     

Tauto Merism and Ionization of Xanthosine

Abstract

Tautomerism and ionization of xanthosine have been studied by infrared spectroscopy. N-methyl and O-methyl model compounds which are isoelectronic with possible keto and enol tautomers have been synthesized. Comparisons of their spectra with neutral and with ionized xanthosine demonstrate that unionized xanthosine has the diketo form and that on acid dissociation (pK 5.7), the first proton is lost from N3 (rather than N1) to give the 6-keto-2-enolate anion. Specific labelling at the 2- and 6-positions with ¹⁸O confirms these conclusions and supports assignment of the highest frequency bands in the double bond region to carbonyl stretching vibrations. These conclusions are relevant to two different proposed structures for the ordered form of polyxanthylic acid (pH 7), one based upon keto and one upon enol tautomeric forms.     

Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways

The 6-oxopurine xanthine (Xan, neutral form 2,6-diketopurine) differs from the corresponding 6-oxopurines guanine (Gua) and hypoxanthine (Hyp) in that, at physiological pH, it consists of a approximately 1:1 equilibrium mixture of the neutral and monoanionic forms, the latter due to ionization of N(3)-H, in striking contrast to dissociation of the N(1)-H in both Gua and Hyp at higher pH. In xanthosine (Xao) and its nucleotides the xanthine ring is predominantly, or exclusively, a similar monoanion at physiological pH. The foregoing has, somewhat surprisingly, been widely overlooked in studies on the properties of these compounds in various enzyme systems and metabolic pathways, including, amongst others, xanthine oxidase, purine phosphoribosyltransferases, IMP dehydrogenases, purine nucleoside phosphorylases, nucleoside hydrolases, the enzymes involved in the biosynthesis of caffeine, the development of xanthine nucleotide-directed G proteins, the pharmacological properties of alkylxanthines. We here review the acid/base properties of xanthine, its nucleosides and nucleotides, their N-alkyl derivatives and other analogues, and their relevance to studies on the foregoing. Included also is a survey of the pH-dependent helical forms of polyxanthylic acid, poly(X), its ability to form helical complexes with a broad range of other synthetic homopolynucleotides, the base pairing properties of xanthine in synthetic oligonucleotides, and in damaged DNA, as well as enzymes involved in circumventing the existence of xanthine in natural DNA.     

Proton release from flavoprotein D-amino acid oxidase on complexation with the zwitterionic ligand, trigonelline

Trigonelline, i.e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.     

Aromaticity balance, π-electron cooperativity and H-bonding properties in tautomerism of salicylideneaniline: the quantum theory of atoms in molecules (QTAIM) approach

Topological analysis based on DFT calculations regarding proton transfer reaction in salicylideneaniline (SA) was performed to scrutinize possible changes in the intramolecular H-bond, π-electron delocalization and aromaticity levels of certain fragments. Quantum chemical calculations and natural bond orbital (NBO) analyses were carried out over a tautomeric ensemble whose members correspond to the molecules at different stages in tautomeric interconversion of SA. The elaboration of intramolecular hydrogen bonding in terms of the relevant topological parameters and the interpretation of certain dependencies regarding its strength were examined. The results show that delocalization index (DI) between donor and acceptor atom δ(O,N) is a useful topological parameter for describing H-bond strength, which is influenced by π-delocalization level within quasiaromatic chelate ring, indicating its resonance-assisted character. NBO analyses reveal that lone-pair (LP) population on N center also affects the strength of intramolecular H-bond in SA. Furthermore, π-electron transfer accompanying intramolecular proton migration in SA is brought into being through formally vacant non-Lewis type LP* orbital on the tautomeric proton. As a result of this, tautomeric protons in molecular entities near TS have hypovalent character due to the lack of electron population in the bonding orbital relative to that in LP* orbital. While H-bonds in the tautomeric ensemble of SA are predominantly partial covalent, molecular entities close to transition state have the strongest covalent H-bonds. The most important result is also that there are linear correlations between the orders of bonds (hydroxyl and amine) involving intramolecular H-bond and electron density values at the relevant BCPs due to partially covalent character of these bonds, contrary to exponential behavior as for purely covalent bonds. Quasiaromatic chelate ring formation is established not only to compel a reduced aromaticity of salicylidene ring but also to decrease in LP-population on N.     

Acceleration of proton exchange between octanol and water by 2,4-dinitrophenol determined by 1H-NMR spectrometry a new model for proton transfer in a hydrophobic environment

The rate of proton transfer between the octanol -OH group and water dissolved in octanol after partition equilibrium was determined by ¹H-NMR spectrometry. The rate was found to depend on the pH of the aqueous phase, being minimal at about pH 11. The uncoupler of oxidative phosphorylation 2,4-dinitrophenol at about 10^?3 M accelerated proton transfer several-fold. Its effect was shown to depend on the concentration of the neutral form of 2,4-dinitrophenol in the octanol phase, irrespective of the pH of the aqueous phase. This effect is suggested to be based on the catalytic action of the phenolic -OH group in 2,4-dinitrophenol. The importance of this effect in the uncoupling action of 2,4-dinitrophenol is discussed.     

Interactions of calf spleen purine nucleoside phosphorylase with 8-azaguanine, and a bisubstrate analogue inhibitor: implications for the reaction mechanism

Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)-8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa approximately 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.     

Photophysical properties of dibenzofluorescein and the presence of its tautomers or prototropic forms in organic solvents.

The UV/Vis absorption and fluorescence properties of dibenzofluorescein (DBFL) in organic solvents were measured and used to shed light on the possible presence of its tautomers or various prototropic forms. DBFL in aprotic solvents mainly exists in two tautomeric forms, viz. quinoid and lactone, but neither are efficiently fluorescent. In protic solvents, such as methanol and ethanol, both the monoanion and neutral quinoid are present and showed the highest fluorescence quantum yield. In contrast, DBFL is fully dissociated to the monoanion and dianion in deionized water.     

Proton-transfer kinetics of photoexcited 7-hydroxy-1-naphthalenesulphonic acid in aqueous DMSO solutions.

The monoanion of 7-hydroxy-1-naphthalenesulphonic acid (HNS) undergoes pseudo-first-order dissociation and its conjugate base undergoes second-order protonation in the lowest excited singlet state. The proton transfer kinetics in water containing dimethylsulphoxide (DMSO), up to a mole fraction of about 0.4, have been evaluated as a function of DMSO concentration. At mole fractions above 0.5 of DMSO, proton-transfer does not measurably occur. At mole fractions below 0.5, steady-state and pulsed-source fluorimetries show the rate constant for dissociation to decrease exponentially with increasing mole fraction of DMSO. This is believed to be due to penetration and disruption of the aqueous solvent cage of HNS by DMSO, resulting in impairment of the Grotthus proton-transfer mechanism. The rate of neutralization of the conjugate base by hydrogen ion is found to vary only slightly with solvent composition and depends on the bulk dielectric properties of the solvent.     

Consequences of one-electron oxidation and one-electron reduction for 4-aminopyrimidine—DFT studies

The consequences of one-electron oxidation and one-electron reduction were studied for 4-aminopyrimidine (4APM), which displays prototropic tautomerism. Since experimental techniques are incapable of detecting less than 0.1% of minor tautomers, quantum-chemical calculations [DFT(B3LYP)/6-311+G(d,p)] were carried out for all possible tautomers of neutral 4AMP and its redox forms, 4APM (+ ?) and 4APM (- ?). Four tautomers were considered: one amine and three imine tautomers (two NH and one CH form). Geometric isomerism of the exo?=?NH group was also taken into account. One-electron oxidation (4APM - e → 4APM (+??)) has no significant effect on the tautomeric preferences; it influences solely the composition of the tautomeric mixture. The amine tautomer is favored for both 4APM (+??) and 4APM. An interesting change in the tautomeric preference occurs for 4APM (- ?). One-electron reduction (4APM?+?e → 4APM (- ?)) favors the C5 atom for the labile proton. The preference of the imine CH tautomer in the tautomeric mixture of 4APM (- ?) may partially explain the origin of CH tautomers in nucleobases.     

Computational study of proton and methyl cation affinities of imidazole-based highly energetic ionic liquids

The present study deals with the evaluation of gas phase proton and methyl cation affinities for alkyl- and nitrosubstituted imidazoles using DFT (B3LYP)/6-31 + G(d) and MP2 methods in the Gaussian 03 software package. The extent of charge delocalization of these cations is correlated with proton affinity. The study reveals that weakly electron-donating alkyl groups at position 1 of the imidazole enhance its proton affinity, which also increases with increasing alkyl chain length. This is expected to result in an increased tendency to form salts. In contrast, the presence of strongly electron-withdrawing nitro groups lowers proton affinity, which decreases as the number of nitro groups on the ring increases. The same trend is observed for the methyl cation affinity, but to a lower degree. These trends in the proton and methyl cation affinities were analyzed to study the effects of these substituents on the basicity of the energetic imidazole moieties and their tendency to form salts. This, in turn, should aid searches for better highly energetic ionic liquids. In addition, calculations performed on different isomers of mono and dinitroimidazoles show that 5-nitro-1H-imidazole and 2,4-dinitro-1H-imidazole are more stable than the other isomers. Amongst the many nitro derivatives of imidazoles considered in the present study, cations resulting from these two would be the best choice for creating highly energetic ionic liquids when coupled with appropriate energetic anions.     

Deamination features of 5-hydroxymethylcytosine,a radical and enzymatic DNA oxidation product

Geometric consequences of electron delocalization were studied for all possible adenine tautomers in aqueous solution by means of ab initio methods {PCM(water)//DFT(B3LYP)/6-311+G(d,p)} and compared to those in the gas phase {DFT(B3LYP)/6-311+G(d,p)}. To measure the consequences of any type of resonance conjugation (π-π, n-π, and σ-π), the geometry-based harmonic oscillator model of electron delocalization (HOMED) index, recently extended to the isolated (DFT) and hydrated (PCM//DFT) molecules, was applied to the molecular fragments (imidazole, pyrimidine, 4-aminopyrimidine, and purine) and also to the whole tautomeric system. For individual tautomers, the resonance conjugations and consequently the bond lengths strongly depend on the position of the labile protons. The HOMED indices are larger for tautomers (or their fragments) possessing the labile proton(s) at the N rather than C atom. Solvent interactions with adenine tautomers slightly increase the resonance conjugations. Consequently, they slightly shorten the single bonds and lengthen the double bonds. When going from the gas phase to water solution, the HOMED indices increase (by less than 0.15 units). There is a good relation between the HOMED indices estimated in water solution and those in the gas phase for the neutral and ionized forms of adenine. Subtle effects, being a consequence of intramolecular interactions between the neighboring groups, are so strongly reduced by solvent that the relation between the HOMED indices and the relative energies for the neutral adenine tautomers seems to be better in water solution than in the gas phase.

Substrate recognition and activation mechanism of D-amino acid oxidase: a study using substrate analogs

We investigated the mechanism of recognition and activation of substrate by D-amino acid oxidase (DAO) by thermodynamical and spectrophotometric methods using zwitterionic ligands [N-methylisonicotinate (NMIN), trigonelline, and homarine] and monoanionic ligands as model compounds of the substrate and the product. In terms of the charge within the substrate D-amino acid, monoanionic (e.g., benzoate), zwitterionic (e.g., NMIN), and dianionic (e.g., terephthalate) ligands are thought to be good models for neutral, basic, and acidic amino acids, respectively, because when a substrate binds to DAO, as previously reported, the a-ammonium group (-NH(3)(+)) probably loses a proton to become neutral (-NH(2)) before the oxidation. Zwitterionic ligands can also be good model compounds of product in the purple complex (the complex of reduced DAO with the product imino acid), because the imino nitrogen of the imino acid is in a protonated cationic form. We also discuss electrostatic interaction, steric effect, and charge-transfer interaction as factors which affect the affinity of substrate/ligand for DAO. Monoanionic ligands have high affinity for neutral forms of oxidized and semiquinoid DAO, while zwitterionic ligands have high affinity for anionic forms of oxidized, semiquinoid, and reduced DAO; this difference was explained by the electrostatic interaction in the active site. The low affinity of homarine (N-methylpicolinate) for oxidized DAO, as in the case of o-methylbenzoate, is due to steric hindrance: one of the ortho carbons of benzoate is near the phenol carbons of Tyr228 and the other ortho carbon is near the carbonyl oxygen of Gly313. The correlation of the affinity of meta- and para-substituted benzoates for oxidized DAO with their Hammet's s values are explained by the HOMO-LUMO interaction between the phenol group of Tyr224 and the benzene ring of benzoate derivative. The pK(a) of neutral flavin [N(3)-H of oxidized flavin, N(5)-H of semiquinoid flavin, and N(1)-H of reduced flavin] decreases by its binding to the apoenzyme. The magnitude of the decrement is oxidized flavin < semiquinoid flavin < reduced flavin. The largest factor in the substantially low pK(a) of reduced flavin in DAO is probably the steric hindrance between the hydrogen atom of H-N(1)(flavin) and the hydrogen atom of H-N of Gly315, which becomes significant when a hydrogen is bound to N(1) of flavin.     

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.     

Conformational preferences of hypermodified nucleoside lysidine (k2C) occurring at "wobble" position in anticodon loop of tRNA(Ile)

Conformational preferences of hypermodified nucleoside, 4-amino-2-(N(6)-lysino)-1-(beta-D-ribofuranosyl) pyrimidinium (Lysidine or 2-lysyl cytidine), usually designated as k(2)C, have been investigated theoretically by the quantum chemical perturbative configuration interaction with localized orbitals (PCILO) method. The zwitterionic, non-zwitterionic, neutral, and tautomeric forms have been studied. Automated geometry optimization using molecular mechanics force field (MMFF), semi-empirical quantum chemical PM3, and ab initio molecular orbital Hartree-Fock SCF quantum mechanical calculations have also been made to compare the salient features. The predicted most stable conformations of zwitterionic, non-zwitterionic, neutral, and tautomeric form are such that in each of these molecules the orientation of lysidine moiety (R) is trans to the N(1) of cytidine. The preferred base orientation is anti (chi = 3 degrees ) and the lysine substituent folds back toward the ribose ring. This results in hydrogen bonding between the carboxyl oxygen O(12a) of lysine moiety and the 2'-hydroxyl group of ribose sugar. In all these four forms of lysidine O(12a)...H-C(9) and O(12b)...H-N(11) interactions provide stability to respective stable conformers. Watson-Crick base pairing of lysidine with A is feasible only with the tautomeric form of usual anti oriented lysidine. This can help in recognition of AUA codon besides in avoiding misrecognition of AUG.     

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.     

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.     

A new model for the agonistic binding site on the histamine H2-receptor: The catalytic triad in serine proteases as a model for the binding site of

The historical model for the agonistic binding site on the histamine H₂-receptor is based on a postulated activation mechanism: it has been suggested that the histamine monocation binds to the histamine H₂-receptor via the formation of three hydrogen bonds. The cationic ammonium group in the side chain and the —NH— group in the π-position of the imidazole act as proton donors, whereas the N— atom in the π-position of the imidazole acts as a proton acceptor. Participation of the ammonium group in H-bonding with a presumed negative charge on the receptor leads to a decrease in positive charge, which is thought to induce a tautomeric change in the imidazole ring system from N^τ-H to N^π-H. A consequence of this tautomeric shift is the donation of a proton from the receptor to the agonist on one side, while on the other side a proton is donated from the agonist to the receptor. The proposed tautomeric shift has been suggested to trigger the H₂-stimulating effect.However, this model for the constitution of the agonistic binding site and the accessory activation mechanism cannot explain the weak histamine H₂-activity of β-histine and the activity of several other recently synthesized H₂-agonists. Based on a thorough literature study and with the aid of molecular electrostatic potentials (MEPs) we demonstrate that the sulphur atom present in histamine H₂-agonists as dimaprit and 2-amino-5-(2-aminoethyl)thiazole does not function as a proton acceptor, which implicitly means that a tautomeric shift is not a prerequisite for H₂-stimulation. As a consequence, the model for the agonistic binding site is adjusted, resulting in a strong resemblance to the nature and orientation of the amino acids constituting the catalytic triad in serine proteases. Within this concept, the N^π-H tautomer of histamine is the biologically active form, in contrast with the existing model in which the N^τ-H tautomer is the active form.     

Hydroxylamine and methoxyamine mutagenesis: tautomeric equilibrium of the promutagenic, N6-methoxyadenosine in solvents of different polarities

Ultraviolet (UV) and Infrared (IR) spectroscopy have been applied to a study of the tautomeric equilibrium, in solvents of varying polarities and differing hydrogen bond donor-acceptor properties, of the promutagenic analogue N6- methoxyadenosine , the product of the reaction of the mutagen methoxyamine with adenosine. In the non-polar solvent CCl4, the analogue is predominantly in the amino form, with KT approximately 10. On transfer from CCl4 to chloroform, dimethyl sulfoxide, and water, the equilibrium is shifted stepwise towards the imino form, attaining a KT approximately 10 in favour of the imino species in aqueous medium. Both the UV and, particularly, the IR spectra exhibit two sets of absorption bands which were assigned to the respective tautomers , in dynamic equilibrium with each other. The significance of the foregoing results in the mechanism of hydroxylamine (and methoxyamine) mutagenesis is considered. It is also shown that base pairing of each tautomeric species is significantly dependent on the conformation of the exocyclic N6-methoxy group. It is further demonstrated that infrared spectroscopy provides data which both supplement and extend those obtained by NMR spectroscopy, under conditions where application of the latter is technically limited.