Tautomeric States Research Articles

Solid state tautomerism in 2-((phenylimino)methyl)naphthalene-1-ol

The solid state tautomerism of 2-((phenylimino)methyl)naphthalene-1-ol was studied using X-ray measurements and absorption spectroscopy. In the solid state, the keto tautomer predominates. The observed shift in the equilibrium from the enol (dilute solution) to the keto (solid state) forms is explained by the formation of dye aggregates using ab initio quantum chemical calculations.

Reaction mechanisms of thiamin diphosphate enzymes

Thiamin diphosphate (ThDP), the biologically active form of vitamin B1, is an essential cofactor that is involved in numerous metabolic pathways, such as the oxidative and non-oxidative decarboxylation of 2-oxo acids, interketol transfer between sugar phosphates, electron transfer reactions and the formation of amino acid precursors. ThDP consists of a thiazolium nucleus, an aminopyrimidine ring, and a diphosphate moiety that is required to provide tight binding to the target enzyme. The electrophilic nature of the positivelycharged thiazolium ring is key to the stabilization of carbanion states of the cofactor (i.e. the ThDP ylide) and of covalent substrate-ThDP conjugates in terms of a ‘Umpolung – or polarity inversion’ mechanism predestining ThDP as an effective participant in the reversible catalytic cleavage of carbon-carbon bonds of vicinal dicarbonyl or a-hydroxyketone groups, exhibiting a chemical ability not possessed by protein functional groups. The first committed step in all ThDP-dependent enzymes is the deprotonation of the thiazolium C2 and attack of the resulting carbanion on a substrate carbonyl. X-ray crystallographic analysis of ThDP enzymes has demonstrated the cofactor to be bound in the canonical V conformation, with the amino group of the aminopyrimidine and C2 of the thiazolium moiety juxtaposed with respect to hydrogen-bonding distance. A strictly conserved glutamate residue interacts with nitrogen 1 of the aminopyrimidine. The conserved structure of the cofactor binding site, and the observed loss of activity accompanyingmutagenesis of the conserved glutamate or uponuse of ThDP analogs lacking nitrogen 1 or the amino group of the aminopyrimidine, all support a mechanism in which proton removal fromC2 of ThDP is assisted by a catalytic triad consisting of the conserved glutamate, the aminopyrimidine and thiazolium moieties of the cofactor, presumably involving the imino tautomer of the aminopyrimidineas thegeneralbase forprotonabstraction. This minireview series, based on lectures given at an international conference on thiamin held in Wittenberg, Germany, in May 2008, highlights recent advancements in the mechanistic understanding of ThDP-promoted biochemical reactions. Jordan et al. have accumulated spectroscopic evidence for the occurrence and interconversion of the different tautomeric and ionization states of enzymebound ThDP along the pathways of various ThDP enzymes. The data clearly implicate the delicately balanced equilibria of the different cofactor forms on the enzymes’ active sites, such that all of these states are thermodynamically accessible at the pH optimum of a given ThDP enzyme. As outlined above, a glutamate residue interacting with the cofactor aminopyrimidine has hitherto been believed to be vital for tautomerization of the aminopyrimidine and thus chemical activation of ThDP. However, structural and mechanistic studies on the ThDP-dependent enzyme glyoxylate carboligase (GCL), as reviewed by Shaanan and Chipman, have revealed valine as having replaced the expected glutamate. Mechanistic analysis suggested that GCL sacrifices the otherwise strictly conserved mechanism of protein-cofactor activation as a consequence of evolutionary adaption to avoid formation of overlystabilized intermediates as thermodynamic traps. The oxidative decarboxylation of 2-oxo acids such as pyruvate is a key reaction of intermediary metabolism in virtually all organisms and requires ThDP to act in tandem with additional cofactors (e.g. flavins, iron-sulfur cluster, lipoic acid) as redox partners. The review by Tittmann, devoted to the mechanistic analysis of redox reactions of ThDP enzymes, shows how some of these enzymes make use of elegant radical chemistry in an intimate coupling mechanism of oxidation–reduction and the formation of energy-rich metabolites such as acetyl-CoA or acetyl phosphate.

Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA – Implications for the catalytic mechanism of parvulins

BackgroundStaphylococcus aureus is a Gram-positive pathogenic bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. Recent emergence of S. aureus strains resistant to numerous antibiotics has created a need for new antimicrobial agents and novel drug targets. S. aureus PrsA is a membrane associated extra-cytoplasmic lipoprotein which contains a parvulin-type peptidyl-prolyl cis-trans isomerase domain. PrsA is known to act as an essential folding factor for secreted proteins in Gram-positive bacteria and thus it is a potential target for antimicrobial drugs against S. aureus.ResultsWe have solved a high-resolution solution structure of the parvulin-type peptidyl-prolyl cis-trans isomerase domain of S. aureus PrsA (PrsA-PPIase). The results of substrate peptide titrations pinpoint the active site and demonstrate the substrate preference of the enzyme. With detailed NMR spectroscopic investigation of the orientation and tautomeric state of the active site histidines we are able to give further insight into the structure of the catalytic site. NMR relaxation analysis gives information on the dynamic behaviour of PrsA-PPIase.ConclusionDetailed structural description of the S. aureus PrsA-PPIase lays the foundation for structure-based design of enzyme inhibitors. The structure resembles hPin1-type parvulins both structurally and regarding substrate preference. Even though a wealth of structural data is available on parvulins, the catalytic mechanism has yet to be resolved. The structure of S. aureus PrsA-PPIase and our findings on the role of the conserved active site histidines help in designing further experiments to solve the detailed catalytic mechanism.

Thymine relaxation after UV irradiation: the role of tautomerization and πσ* states

Ab initio calculations and femtosecond pump-probe ionization experiments were carried out to identify excited state relaxation processes in isolated thymine monomer and small thymine-water clusters. Three transient species with life times of < or =100 fs, 7 ps and >1 ns were observed in the experiments on gas phase thymine. The longer-lived transients were weak or absent in thymine-water clusters. Available theoretical results on thymine agree with the assignment of low-lying pi-pi* and n-pi* excited states to the femtosecond and picosecond transients but the assignment of the third transient remains opaque. Our theoretical results seem to exclude the possibility of ground or excited state tautomerization as well as the involvement of states with pi-sigma* character. Remaining explanations for the observed transients are: very fast intersystem crossing to the triplet manifold or the observation of transient signals from local minima on the potential energy surfaces.

Transverse relaxation-optimized HCN experiment for tautomeric states of histidine sidechains

Function of protein is frequently related with tautomeric states of histidine sidechains. Thus, several NMR experiments were developed to determine the tautomeric states of histidines. However, poor sensitivity of these experiments caused by long duration of magnetization transfer periods is unavoidable. Here, we alleviate the sensitivity of HCN experiment for determining the tautomeric states of histidine residues using TROSY principle to suppress transverse relaxation of <TEX>$^{13}C$</TEX> spins during long polarization transfer delays involving <TEX>$^{13}C-^{15}N$</TEX> scalar couplings. In addition, this experiment was used to assign the sidechain resonances of histidines. These assignments can be used to follow the pH-titration of histidine sidechains.

Solution structure of NPr, a bacterial signal-transducing protein that controls the phosphorylation state of the potassium transporter-regulating protein IIANtr

A nitrogen-related signal transduction pathway, consisting of the three phosphotransfer proteins EI(Ntr), NPr, and IIA(Ntr), was discovered recently to regulate the uptake of K(+) in Escherichia coli. In particular, dephosphorylated IIA(Ntr) inhibits the activity of the K(+) transporter TrkA. Since the phosphorylation state of IIA(Ntr) is partially determined by its reversible phosphorylation by NPr, we have determined the three-dimensional structure of NPr by solution NMR spectroscopy. In total, we obtained 973 NOE-derived distance restraints, 112 chemical shift-derived backbone angle restraints, and 35 hydrogen-bond restraints derived from temperature coefficients (wave). We propose that temperature wave is useful for identifying exposed beta-strands and assists in establishing protein folds based on chemical shifts. The deduced structure of NPr contains three alpha-helices and four beta-strands with the three helices all packed on the same face of the beta-sheet. The active site residue His16 of NPr for phosphoryl transfer was found to be neutral and in the N epsilon 2-H tautomeric state. There appears to be increased motion in the active site region of NPr compared to HPr, a homologous protein involved in the uptake and regulation of carbohydrate utilization.

Electrostatic Effects in a Network of Polar and Ionizable Groups in Staphylococcal Nuclease

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the p K a values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with p K a values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the p K a values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a p K a was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N ε2 state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based p K a calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where p K a values are governed by short-range polar and Coulomb interactions.

Assignment of protonation states and geometries to macromolecular structures using Unary Quadratic Optimization

A method is presented for assigning protonation states and hydrogen atom geometry to macromolecular structures. Tautomer and ionization state (including metal ligation) are taken into account during a large scale free energy optimization. This discrete optimization is performed with a Unary Quadratic Optimization procedure. The thermodynamic theory and the results of several computational validation experiments will be presented.

Tautomerism in structure-based 3D pharmacophore modeling

Tautomeric rearrangements on molecules lead to distinct equilibrated structural states of the same chemical compound, and evidently, have an impact on nearly all aspects of computer-aided chemical data processing [1] where the knowledge of the exact chemical structure is required (e.g. the calculation of chemical properties or interpretation of ligand-protein interactions). Although tautomerism is a well-known and well-documented phenomenon, it has long been disregarded in the field of drug design, which, as a consequence, leads to a possible misinterpretation of ligand binding interactions. Especially heuristic approaches, like structure-based pharmacophore modeling, are techniques that allow for consideration of several tautomeric forms, although this has not been done systematically by now. Recent approaches in the area of drug design mainly focus on virtual screening and tautomer enrichment of existing compound databases [2]. However, the effect of tautomerism in deriving interaction patterns within the protein-ligand complex (structure-based 3D pharmacophore modeling) has not yet been sufficiently addressed and thus leaves a wide and interesting field for future investigations. In the presented work various protein-ligand complexes have been studied where the tautomeric states of the ligand have a significant impact on derived 3D pharmacophores and the perception of binding modes, and also, tautomer-invariant pharmacophore patterns are introduced. Furthermore, an algorithm will be presented which enumerates all possible tautomers on both the receptor and ligand side under the constraints of a fixed active ligand conformation.

Crystal structures of Toxoplasma gondii pterin-4a-carbinolamine dehydratase and comparisons with mammalian and parasite orthologues

The enzyme pterin-4a-carbinolamine dehydratase (PCD) is important for the recycling of pterins within eukaryotic cells. A recombinant expression system for PCD from the apicomplexan parasite Toxoplasma gondii has been prepared, the protein purified and crystallised. Single crystal X-ray diffraction methods have produced a high-resolution structure (1.6 Å) of the apo-enzyme and a low-resolution structure (3.1 Å) of a complex with a substrate-like ligand dihydrobiopterin (BH 2). Analysis of the hydrogen bonding interactions that contribute to binding BH 2 suggest that the ligand is present in an enol tautomeric state, which makes it more similar to the physiological substrate. The enzyme can process ( R)- and ( S)-forms of pterin-4a-carbinolamine and the ligand complex suggests that His61 and His79 are placed to act independently as general bases for catalysis of the individual enantiomers. Comparisons with orthologues from other protozoan parasites ( Plasmodium falciparum and Leishmania major) and with rat PCD, for which the structure is known, indicate a high degree of sequence and structure conservation of this enzyme. The molecular determinants of ligand recognition and PCD reactivity are therefore highly conserved across species.

Epik: a software program for pK a prediction and protonation state generation for drug-like molecules

Epik is a computer program for predicting pK(a) values for drug-like molecules. Epik can use this capability in combination with technology for tautomerization to adjust the protonation state of small drug-like molecules to automatically generate one or more of the most probable forms for use in further molecular modeling studies. Many medicinal chemicals can exchange protons with their environment, resulting in various ionization and tautomeric states, collectively known as protonation states. The protonation state of a drug can affect its solubility and membrane permeability. In modeling, the protonation state of a ligand will also affect which conformations are predicted for the molecule, as well as predictions for binding modes and ligand affinities based upon protein-ligand interactions. Despite the importance of the protonation state, many databases of candidate molecules used in drug development do not store reliable information on the most probable protonation states. Epik is sufficiently rapid and accurate to process large databases of drug-like molecules to provide this information. Several new technologies are employed. Extensions to the well-established Hammett and Taft approaches are used for pK(a) prediction, namely, mesomer standardization, charge cancellation, and charge spreading to make the predicted results reflect the nature of the molecule itself rather just for the particular Lewis structure used on input. In addition, a new iterative technology for generating, ranking and culling the generated protonation states is employed.

Elucidation of the Chemistry of Enzyme-Bound Thiamin Diphosphate Prior to Substrate Binding: Defining Internal Equilibria among Tautomeric and Ionization States

Both solution and crystallographic studies suggest that the 4'-aminopyrimidine ring of the thiamin diphosphate coenzyme participates in catalysis, likely as an intramolecular general acid-base catalyst via the unusual 1',4'-iminopyrimidine tautomer. It is indeed uncommon for a coenzyme to be identified in its rare tautomeric form on its reaction pathways, yet this has been possible with thiamin diphosphate, in some cases even in the absence of substrate [Nemeria, N., Chakraborty, S., Baykal, A., Korotchkina, L., Patel, M. S., and Jordan, F. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 78-82.]. The ability to detect both the aminopyrimidine and iminopyrimidine tautomeric forms of thiamin diphosphate on enzymes has enabled us to assign the predominant tautomeric form present in individual intermediates on the pathway. Herein, we report the pH dependence of these tautomeric forms providing the first data for the internal thermodynamic equilibria on thiamin diphosphate enzymes for the various ionization and tautomeric forms of this coenzyme on four enzymes: benzaldehyde lyase, benzoylformate decarboxylase, pyruvate oxidase, and the E1 component of the human pyruvate dehydrogenase multienzyme complex. Evidence is provided for an important function of the enzyme environment in altering both the ionization and tautomeric equilibria on the coenzyme even prior to addition of substrate. The pKa for the 4'-aminopyrimidinium moiety coincides with the pH for optimum activity thereby ensuring that all ionization states and tautomeric states are accessible during the catalytic cycle. The dramatic influence of the protein on the internal equilibria also points to conditions under which the long-elusive ylide intermediate could be stabilized.

Probing the potential energy landscape for dissociation of protonated indole via threshold collision-induced dissociation and theoretical studies

Collision-induced dissociation of protonated indole with Xe is studied as a function of kinetic energy using guided ion beam tandem mass spectrometry techniques. Activated dissociation resulting in endothermic loss of neutral HCN (or HNC) is the only pathway observed over the range of collision energies examined. The cross-section threshold for this activated dissociation pathway is interpreted to yield 0 and 298 K activation energies for this process after accounting for the effects of multiple ion–neutral collisions, the internal energy distribution of the protonated indole cations, and their lifetimes for dissociation. Density functional theory (DFT) calculations at the MPW1PW91/6-31G* level of theory are used to determine the structures of indole, the protonated indole tautomers, and the transition states, intermediates, and products involved in the activated dissociation of protonated indole. Four distinct pathways between the reactant ion and dissociation products are computed. In all cases, the ionic product formed is C 6H 5CH 2 +, while the neutral product is HCN in three of the pathways and HNC in the fourth. The vibrational frequencies and rotational constants of the ground state tautomer of protonated indole and the rate-determining transition state along each of the pathways computed are used for the thermodynamic analysis of the experimental data. The theoretical activation energies and potential energy landscapes for activated dissociation of protonated indole are determined from single point calculations at the MPW1PW91/6-311+G(2d,2p) and MP2(full)/6-311+G(2d,2p) levels of theory, using the MPW1PW91/6-31G* optimized geometries. Both theories produce similar potential energy landscapes for elimination of HCN (or HNC) from protonated indole. Theory suggests that elimination of HNC is favored over HCN. However, our threshold measurements probe the lowest-energy pathway available and are in much better agreement with the higher-energy HCN elimination pathways, suggesting that theory underestimates the activation energy for loss of HNC.

Intramolecular Proton-Transfer Processes Starting at Higher Excited States: A Fluorescence Study on 2-Butylamino-6-methyl-4-nitropyridine N-Oxide in Nonpolar Solutions

This article describes the exceptional photophysics of 2-butylamino-6-methyl-4-nitropyridine N-oxide (2B6M). It is known from the literature that this compound may undergo excited-state intra- or intermolecular proton-transfer reactions. In nonpolar solvents, 2B6M exhibits an unusual fluorescence behavior: there is a substantial difference between the relative band intensities of the excitation and absorption spectra. Furthermore, in emission two bands are observed, and their relative intensities depend on the excitation wavelength, thus violating the Kasha-Vavilov rule. It is the objective of this research to interpret these results. For this purpose, steady-state fluorescence excitation and emission spectra in the liquid state were recorded and quantum yields were determined for the two types of emission. In addition, absorption spectra were measured at room temperature and under low-temperature conditions. Finally, fluorescence lifetimes of the emitting species were determined using the time-correlated single photon counting technique. The results suggest that in the liquid state only one (monomeric) ground state species dominates, which can emit via two different pathways (from the normal and the tautomeric excited state). The excitation spectra point at two different internal proton-transfer processes, one starting at the S1 state and one starting at the S2 state. On the basis of the measured lifetimes and fluorescence quantum yields, a kinetic scheme was completed that can quantitatively explain the observations.

Observation by NMR of the tautomerism of an intramolecular OHOHN-charge relay chain in a model Schiff base

As a model system for the internal and external aldimines of the coenzyme pyridoxal phosphate (PLP) in PLP dependent enzymes we have studied the 1H and 15N NMR spectra of the 15N labeled Schiff base 3-carboxy-5-methyl-salicylidenaniline ( 1) dissolved in CD 2Cl 2. 1 contains a charge relay system with two strongly coupled intramolecular hydrogen bonds of the OHOHN type. One-bond 15N 1H scalar spin–spin coupling constants and chemical shifts of partially deuterated 1 were measured in the temperature range between 243 and 183 K and analyzed assuming an exchange between three tautomeric states exhibiting well defined hydrogen bond geometries. The analysis shows that the dominant structure 1b corresponds to the zwitterion O H⋯O −⋯H N +, where deuteration of one bond leads to a shortening of the other. This anti-cooperative effect is revealed by the vicinal isotope effects on the proton chemical shifts. By contrast, forms 1a and 1c are characterized by the structures O H⋯O H⋯N and O −⋯H O⋯H N +, correspondingly, whose hydrogen bonds exhibit a cooperative coupling. We predict that 1a will dominate at high temperatures and low dielectric constants, whereas 1c will dominate at low temperatures and large dielectric constants. The comparison with model systems which do not contain the additional COOH-group indicates that the latter is responsible for the dominance of the zwitterionic structure of the OHN hydrogen bond. The implications of these findings for the function of the coenzyme pyridoxal phosphate in its natural environment are discussed.

Catalytic Mechanism of Guanine Deaminase: An ONIOM and Molecular Dynamics Study

The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 A compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of approximately 8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier approximately 8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.

Synthesis of π-Conjugated Ferrocene-Anthraquinone Alternating Polymers and their Protonation Reactions

The ethynylene-bridged ferrocene-anthraquinone system exhibits unique protonation-induced electron transfer reactions to form multi-dentate valence tautomerization states. In this study, we newly synthesized ethynylene-bridged ferrocene-anthraquinone alternating oligomers by a reaction of 1,1′-bis(trimethylstannylethynyl)ferrocene with 1,8-dibromoanthraquinone in the presence of a Pd(0) catalyst. An insoluble 1:1-donor/acceptor polymer, (1,1′-Fc-1,8-Aq)n, was obtained and characterized by IR and UV-vis spectra and elemental analysis. The molecular weight of the soluble portion of the oligomers was Mw = 2.6 × 103 (Mw/Mn = 1.4). A cyclic dimer, cyclic-(FcAq)2, was also obtained and characterized by the 2D-NMR spectra. The behavior of the insoluble polymer (1,1′-Fc-1,8-Aq)n upon the addition of an organic acid such as CF3SO3H was examined and compared with that of the monomer exposed to the same conditions. The addition of CF3SO3H to the insoluble polymer rendered soluble in organic solvent, and the changes in the UV-vis-NIR and IR absorption spectra reflected the structural changes to the protonated form with valence tautomers; this reaction was similar to the reactions with 1-ferrocenylethynylanthraquinone (1-FcAq) and 1,8-bis(ferrocenylethynyl)anthraquinone (1,8-Fc2Aq).

Fluorescent dyes undergoing intramolecular proton transfer with improved sensitivity to surface charge in lipid bilayers

4'-(Dialkylamino)-3-hydroxyflavones are characterized by an excited-state proton transfer reaction between two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Due to the high sensitivity of the relative intensities of the two emission bands to solvent polarity, hydrogen bonding and local electric fields, these dyes found numerous applications in biomembrane studies. In order to further improve their fluorescence characteristics, we have synthesized new dyes where the 2-phenyl group is substituted with a 2-thienyl group. In organic solvents, the new dyes exhibit red shifted absorption and dual fluorescence. Although they show lower sensitivity to solvent polarity and H-bond donor ability (acidicity) than their parent 3-hydroxyflavone dyes, they exhibit a much higher sensitivity to solvent H-bond acceptor ability (basicity). Moreover, when tested in lipid vesicles of different surface charge, the new dyes show much better resolved dual emission and higher sensitivity to the surface charge of lipid bilayers than the parent dyes. The response of the new dyes to surface charge is probably connected with the H-bond basicity of the membrane surface, which is the highest for negatively charged surfaces. As a consequence, the new dyes appear as prospective fluorophores for the development of new fluorescent probes for biomembranes.

Hydrogen bonding in the fructose‐2,6‐bisphosphatase reaction correlates with activity

The bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, and the associated biofactor, fructose-2,6-bisphosphate (F-2,6-P2) are important regulators of carbohydrate metabolism. We have used multi-dimensional NMR spectroscopy to determine the tautomeric and protonation states of the catalytic histidines in the bisphosphatase domain from human heart (hhFBP-2). Resonances were assigned by comparison to those conserved in the rat liver bisphosphatase (rlFBP-2). The FBP-2 reaction utilizes a stable phosphohistidine intermediate that can be observed by NMR spectroscopy. In resting hhFBP-2 the phospho-acceptor, His-257, exists as the neutral N1′ tautomer, the other catalytic residue, His-391, is cationic, as is His-419, while His-445 is in the neutral N3′ tautomeric form. The 13C and 1H chemical shifts assigned to the catalytic histidines in hhFBP-2 are consistently up-field of the corresponding resonances in rlFBP-2, particularly in the transient reaction intermediate. These reveal isozyme-specific changes in the bisphosphatase active site that suggest weaker hydrogen bonds within the cardiac bisphosphatase transient intermediate. Because these hydrogen bonds stabilize the phosphohistidine intermediate, the observed differences can be related to physiologically appropriate differences in the FBP-2 activity in liver and heart.

A CASPT2 study of the excited states of adenine tautomers with silver ions

The multiconfigurational complete active space (CAS) SCF method and multi-state second order perturbation theory (MS-CASPT2) are used to study the electronic spectra of adenine and the seven low-lying energy tautomers of adenine with the metal cation Ag(I). In all metal complexes a weak Rydberg 4d → 5s transition has been found below the two valence 1π → π ∗ states, also identified in the isolated adenine. In most cases, silver metalation leads to a red-shift of the first and a blue-shift of the second valence 1π → π ∗ bands, with respect to the isolated adenine. Energetically the most favourable Ag–adenine species in gas phase are bidentated, however, the experimental spectrum in solution is explained with the presence of monodentated tautomers, suggesting that bidentation is destabilized in aqueous phase.