N1 Proton Research Articles

Conformational stabilities of iminoallantoin and its base pairs in DNA: implications for mutagenicity.

The types of mutations induced by oxidatively damaged products of DNA are continuously in debate. For example, some biochemical studies have proposed that guanidinohydantoin (Gh) would induce exclusively G to C mutations, while other studies have predicted a mixture of various mutations including G to C, G to T and G to A. In addition to the nature of mutations, the exact reasons of these mutations are also not properly understood. It is suggested that Gh can easily isomerize to iminoallantoin (Ia) in a pH-dependent manner and the transition becomes complete at pH > 8. In order to understand Gh/Ia-induced mutations, we have here studied the role of the most stable tautomer of Ia in the R- and S-enantiomeric configurations in promoting mismatch base pair complexes in DNA by employing a density functional theoretical (DFT) approach. It is found that Ia can have 39 different possible tautomeric forms each in the R- and S-enantiomeric configurations, out of which the most stable tautomer would involve the deprotonation of the N1 atom and protonation of the N3 atom. The most stable tautomer of Ia can adopt three different rotameric conformations (Ia1, Ia2, and Ia3) of comparable stabilities. It is further revealed that these rotamers of Ia can interact with different bases of DNA in 88 different possible ways. However, the interaction of G with Ia3 in both the anti- and syn-conformations would be the most stable. It is further revealed that the base pairing patterns, binding energies and electronic environments of anti-Ia3:G and G:T complexes are similar. In addition to this, it is also found that the binding patterns and energies of Gh1:G and Ia3:G complexes are similar. Based on these results, it is proposed that under physiological conditions, Gh1 may be responsible for the observed G to C mutations in DNA, while in an acidic environment Ia3 may be responsible for the same mutations. This study has led to a solid foundation for further high resolution structural studies to completely unravel Ia-induced mutagenicity in DNA.

Mechanisms and energetics for N-glycosidic bond cleavage of protonated adenine nucleosides: N3 protonation induces base rotation and enhances N-glycosidic bond stability.

Our previous gas-phase infrared multiple photon dissociation action spectroscopy study of protonated 2'-deoxyadenosine and adenosine, [dAdo+H](+) and [Ado+H](+), found that both N3 and N1 protonated conformers are populated with the N3 protonated ground-state conformers predominant in the experiments. Therefore, N-glycosidic bond dissociation mechanisms of N3 and N1 protonated [dAdo+H](+) and [Ado+H](+) and the associated quantitative thermochemical values are investigated here using both experimental and theoretical approaches. Threshold collision-induced dissociation (TCID) of [dAdo+H](+) and [Ado+H](+) with Xe is studied using guided ion beam tandem mass spectrometry techniques. For both systems, N-glycosidic bond cleavage reactions are observed as the major dissociation pathways resulting in production of protonated adenine or elimination of neutral adenine. Electronic structure calculations are performed at the B3LYP/6-311+G(d,p) level of theory to probe the potential energy surfaces (PESs) for N-glycosidic bond cleavage of [dAdo+H](+) and [Ado+H](+). Relative energetics of the reactants, transition states, intermediates and products along the PESs for N-glycosidic bond cleavage are determined at the B3LYP/6-311+G(2d,2p), B3LYP-GD3BJ/6-311+G(2d,2p), and MP2(full)/6-311+G(2d,2p) levels of theory. The predicted N-glycosidic bond dissociation mechanisms for the N3 and N1 protonated species differ. Base rotation of the adenine residue enables formation of a strong N3H(+)O5' hydrogen-bonding interaction that stabilizes the N3 protonated species and its glycosidic bond. Comparison between experiment and theory indicates that the N3 protonated species determine the threshold energies, as excellent agreement between the measured and B3LYP computed activation energies (AEs) and reaction enthalpies (ΔHrxns) for N-glycosidic bond cleavage of the N3 protonated species is found.

TEMPO-derived spin labels linked to the nucleobases adenine and cytosine for probing local structural perturbations in DNA by EPR spectroscopy.

Three 2´-deoxynucleosides containing semi-flexible spin labels, namely TA, UA and UC, were prepared and incorporated into deoxyoligonucleotides using the phosphoramidite method. All three nucleosides contain 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) connected to the exocyclic amino group; TA directly and UA as well as UC through a urea linkage. TA and UC showed a minor destabilization of a DNA duplex, as registered by a small decrease in the melting temperature, while UA destabilized the duplex by more than 10 °C. Circular dichroism (CD) measurements indicated that all three labels were accommodated in B-DNA duplex. The mobility of the spin label TA varied with different base-pairing partners in duplex DNA, with the TA•T pair being the least mobile. Furthermore, TA showed decreased mobility under acidic conditions for the sequences TA•C and TA•G, to the extent that the EPR spectrum of the latter became nearly superimposable to that of TA•T. The reduced mobility of the TA•C and TA•G mismatches at pH 5 is consistent with the formation of TAH+•C and TAH+•G, in which protonation of N1 of A allows the formation of an additional hydrogen bond to N3 of C and N7 of G, respectively, with G in a syn-conformation. The urea-based spin labels UA and UC were more mobile than TA, but still showed a minor variation in their EPR spectra when paired with A, G, C or T in a DNA duplex. UA and UC had similar mobility order for the different base pairs, with the lowest mobility when paired with C and the highest when paired with T.

The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.

The role of N7 protonation of guanine in determining the structure, stability and function of RNA base pairs.

The roles of protonated nucleobases in stabilizing different structural motifs and in facilitating catalytic functions of RNA are well known. Among different polar sites of all the nucleobases, N7 of guanine has the highest protonation propensity at physiological pH. However, unlike other easily protonable sites such as N1 and N3 of adenine or N3 of cytosine, N7 protonation of guanine does not lead to the stabilization of base pairs involving its protonated Hoogsteen edge. It also does not facilitate its participation in any acid-base catalysis process. To explore the possible roles of N7 protonated guanine, we have studied its base pairing potentials involving WatsonCrick and sugar edges, which undergo major charge redistribution upon N7 protonation. We have carried out quantum chemical geometry optimization at the M05-2X/6-311G+(2d,2p) level, followed by interaction energy calculation at the MP2/aug-cc-pVDZ level, along with the analysis of the context of occurrence for selected base pairs involving the sugar edge or the WatsonCrick edge of guanine within a non-redundant set of 167 RNA crystal structures. Our results suggest that, four base pairs - G:C W:W trans, G:rC W:S cis, G:G W:H cis and G:G S:H trans may involve N7 protonated guanine. These base pairs deviate significantly from their respective experimental geometries upon QM optimization, but they retain their experimental geometries if guanine N7 protonation is considered during optimization. Our study also reveals the role of guanine N7 protonation (i) in stabilizing important RNA structural motifs, (ii) in providing a framework for designing pH driven molecular motors and (iii) in providing an alternative strategy to mimic the effect of post-transcriptional changes.

Gas-Phase Conformations and Energetics of Protonated 2'-Deoxyguanosine and Guanosine: IRMPD Action Spectroscopy and Theoretical Studies.

The gas-phase structures of protonated 2'-deoxyguanosine, [dGuo+H](+), and its RNA analogue protonated guanosine, [Guo+H](+), are investigated by infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical electronic structure calculations. IRMPD action spectra are measured over the range extending from ∼550 to 1900 cm(-1) using the FELIX free electron laser and from ∼2800 to 3800 cm(-1) using an optical parametric oscillator/amplifier (OPO/OPA) laser system. The measured IRMPD spectra of [dGuo+H](+) and [Guo+H](+) are compared to each other and to B3LYP/6-311+G(d,p) linear IR spectra predicted for the stable low-energy conformations computed for these species to determine the most favorable site of protonation, identify the structures accessed in the experiments, and elucidate the influence of the 2'-hydroxyl substituent on the structures and the IRMPD spectral features. Theoretical energetics and the measured IRMPD spectra find that N7 protonation is preferred for both [dGuo+H](+) and [Guo+H](+), whereas O6 and N3 protonated conformers are found to be much less stable. The 2'-hydroxyl substituent does not exert a significant influence on the structures and relative stabilities of the stable low-energy conformations of [dGuo+H](+) versus [Guo+H](+) but does provide additional opportunities for hydrogen bonding such that more low-energy structures are found for [Guo+H](+). [dGuo+H](+) and [Guo+H](+) share very parallel IRMPD spectral features in the FELIX and OPO regions, whereas the effect of the 2'-hydroxyl substituent is primarily seen in the relative intensities of the measured IR bands. The measured OPO/OPA spectral signatures, primarily reflecting the IR features associated with the O-H and N-H stretches, provide complementary information to that of the FELIX region and enable the conformers that arise from different protonation sites to be more readily distinguished. Insight gained from this and parallel studies of other DNA and RNA nucleosides and nucleotides should help better elucidate the effects of the local environment on the overall structures of DNA and RNA.

Theoretical study on the detailed repair of O6-methyl guanine to guanine by cysteine

The repair process of O6-methylguanine (O6mG) by cysteine (Cys145) has been studied using the quantum mechanical calculations in the presence of solvent water molecules and also using the polarizable continuum model (PCM). The proton transfer from Cys145 to O6mG, directly and mediated by bridged water molecules, has been considered to investigate the role and the sequence of the proton transfer on the repair process. In both models, the energy barrier of the methyl transfer is reduced when the proton is initially transferred from O6mG to Cys145. In addition, the protonation of N3 and N7 sites of O6mG in the acidic environment facilitates the repair process. Results of this study help to understand repair mechanisms of damaged DNA bases better on the basis of proton transfer between that and nucleic acids. The effects of solvent and protonation of O6mG on the energy barrier of the repair process by cysteine have been considered using six models. The results indicate that the repair process is facilitated in the acidic environment.

Crystallographic approach to determination of active conformations of LCAPs

Long-Chain Aryl-Piperazines (LCAPs) are well known serotonin receptor ligands used in several marketed antidepressant drugs. LCAPs consist of three structural units: a terminal group, an arylpiperazine at one N atom and an aliphatic chain (spacer) at the other N atom joining the two former units. Both the arylpiperazine and the terminal groups have rather rigid structures and thus their conformational freedom is limited. The opposite is true for the aliphatic spacer, which allows practically any orientation of the terminal group. The resulting diversity of conformations observed in the crystals of LCAPs is significant, which explains their affinity to many serotonin receptors. There is a vast literature on the subject and some qualitative observations were developed. However, due to the flexible spacer and diversity of the terminal groups, their usefulness is limited. Our X-ray (16 crystal structures) and affinity studies on almost sixty new LCAPs [1], together with the data from CSD, enable us to determine the common conformations of LCAPs and the relationships between structure, affinity and conformation. In the analysis, the following features were considered: (i) - axial/equatorial orientations of the substituents of the piperazine ring; (ii) –N1 protonation possible in the physiological environment; (iii) - a twist of the aryl ring; (iv) –the parity and the number of atoms in the spacer; (v) – the presence of heteroatoms or groups in the spacer; (vi) – the spatial position of the terminal group in relation to the piperazine ring.

Crystal structure of unusual RNA duplex containing strontium ion binding motif

Crystal structures of several functional non-coding RNAs, such as ribozymes, aptamers, ribosomes and tRNAs, have been reported so far. Unusual structural motifs and non-complementary base pairs are important for their functions. In the present study, we have determined a crystal structure of an unusual RNA duplex containing a strontium ion binding motif. A 19 mer RNA (5'-UUGUCGCUU[Br]CGAAAAAGUC-3') was chemical synthesized and purified by denaturing PAGE. Crystallizations were performed by the sitting-drop vapor diffusion method. The initial phase was solved by the SAD method. Atomic parameters were refined at a resolution of 3.0 Å. The 19 mer RNA forms an unusual antiparallel duplex. At both ends of the duplex, the Watson-Crick G=C and A-U and the Wobble GoU and AoC base pairs are formed. The Wobble C10oA14* pair is available only in acidic condition by protonation of N1 of A14* (* indicates residues of the opposite strand). Two hydrogen bonds, N1-H(A14*)...O2(C10) and N6-H(A14*)...N3(C10), are observed in the base pair. In the center of the duplex, two sheared G11oA13* and G11*oA13 base pairs are formed. The distance between two RNA chains becomes shorter by the GoA base pair and hydrogen bonds between the Watson-Crick edge of G11 and the phosphate group of A12*. Therefore, the central A12 residue cannot make a base pair, but it makes a stacking interaction with A12*. The A12 residue stacks also with A13 of the sheared GoA base pair. As a result, an A13-A12-A12*-A13* stacked column is formed at the minor groove of the duplex, and the G11 base of the sheared GoA base pair is inclined toward the minor groove. By taking such a unique structure, the RNA duplex has a Sr2+ ion binding pocket in the center. A hydrated Sr2+ ion binds to O6 and N7 of G11 and G11*. The Sr2+ ion is surrounded by four phosphate groups of two RNA chains. The Sr2+ ion is tightly captured by eight hydrogen bonds in total.

Analysis of guanine oxidation products in double-stranded DNA and proposed guanine oxidation pathways in single-stranded, double-stranded or quadruplex DNA.

Guanine is the most easily oxidized among the four DNA bases, and some guanine-rich sequences can form quadruplex structures. In a previous study using 6-mer DNA d(TGGGGT), which is the shortest oligomer capable of forming quadruplex structures, we demonstrated that guanine oxidation products of quadruplex DNA differ from those of single-stranded DNA. Therefore, the hotooxidation products of double-stranded DNA (dsDNA) may also differ from that of quadruplex or single-stranded DNA, with the difference likely explaining the influence of DNA structures on guanine oxidation pathways. In this study, the guanine oxidation products of the dsDNA d(TGGGGT)/d(ACCCCA) were analyzed using HPLC and electrospray ionization-mass spectrometry (ESI-MS). As a result, the oxidation products in this dsDNA were identified as 2,5-diamino-4H-imidazol-4-one (Iz), 8-oxo-7,8-dihydroguanine (8oxoG), dehydroguanidinohydantoin (Ghox), and guanidinohydantoin (Gh). The major oxidation products in dsDNA were consistent with a combination of each major oxidation product observed in single-stranded and quadruplex DNA. We previously reported that the kinds of the oxidation products in single-stranded or quadruplex DNA depend on the ease of deprotonation of the guanine radical cation (G•+) at the N1 proton. Similarly, this mechanism was also involved in dsDNA. Deprotonation in dsDNA is easier than in quadruplex DNA and more difficult in single-stranded DNA, which can explain the formation of the four oxidation products in dsDNA.

Increasing occurrences and functional roles for high energy purine-pyrimidine base-pairs in nucleic acids

There are a growing number of studies reporting the observation of purine-pyrimidine base-pairs that are seldom observed in unmodified nucleic acids because they entail the loss of energetically favorable interactions or require energetically costly base ionization or tautomerization. These high energy purine-pyrimidine base-pairs include G•C(+) and A•T Hoogsteen base-pairs, which entail ∼180° rotation of the purine base in a Watson-Crick base-pair, protonation of cytosine N3, and constriction of the C1'-C1' distance by ∼2.5Å. Other high energy pure-pyrimidine base-pairs include G•T, G•U, and A•C mispairs that adopt Watson-Crick like geometry through either base ionization or tautomerization. Although difficult to detect and characterize using biophysical methods, high energy purine-pyrimidine base-pairs appear to be more common than once thought. They further expand the structural and functional diversity of canonical and non-canonical nucleic acid base-pairs.

The contribution of pseudouridine to stabilities and structure of RNAs

Thermodynamic data are reported revealing that pseudouridine (Ψ) can stabilize RNA duplexes when replacing U and forming Ψ-A, Ψ-G, Ψ-U and Ψ-C pairs. Stabilization is dependent on type of base pair, position of Ψ within the RNA duplex, and type and orientation of adjacent Watson–Crick pairs. NMR spectra demonstrate that for internal Ψ-A, Ψ-G and Ψ-U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. Ψ has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow Ψ to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of Ψ.

Specific solvent effect on lumazine photophysics: A combined fluorescence and intrinsic reaction coordinate analysis

The photophysical properties and tautomerization behavior of neutral lumazine were studied by fluorescence spectroscopy and density functional theory calculation. A quantitative estimation of the contributions from different solvatochromic parameters, like solvent polarizibility (π∗), hydrogen bond donation (α) and hydrogen bond accepting (β) ability of the solvent, was made using linear free energy relationships based on the Kamlet–Taft equation. The analysis reveals that the hydrogen bond acceptance ability of the solvent is the most important parameter characterizing the excited state behavior of lumazine. Theoretical calculations result predict an extensive charge redistribution of lumazine upon excitation corresponding to the N3 and N1 proton dissociation sites by solvents in the ground and excited states, respectively. Comparison of S0 and S1 state potential energy curves constructed for several water mediated tautomerization processes by intrinsic reaction coordinate analysis of lumazine-H2O cluster shows that (3,2) and (1,8) hydrogen migrations are the most favorable processes upon excitation.

Characterizing the Protonation State of Cytosine in Transient G·C Hoogsteen Base Pairs in Duplex DNA

G·C Hoogsteen base pairs can form transiently in duplex DNA and play important roles in DNA recognition, replication, and repair. G·C Hoogsteen base pairs are thought to be stabilized by protonation of cytosine N3, which affords a second key hydrogen bond, but experimental evidence for this is sparse because the proton cannot be directly visualized by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here, we combine NMR and constant pH molecular dynamics simulations to directly investigate the pKa of cytosine N3 in a chemically trapped N1-methyl-G·C Hoogsteen base pair within duplex DNA. Analysis of NMR chemical shift perturbations and NOESY data as a function of pH revealed that cytosine deprotonation is coupled to a syn-to-anti transition in N1-methyl-G, which results in a distorted Watson-Crick geometry at pH >9. A four-state analysis of the pH titration profiles yields a lower bound pKa estimate of 7.2 ± 0.1 for the G·C Hoogsteen base pair, which is in good agreement with the pKa value (7.1 ± 0.1) calculated independently using constant pH MD simulations. Based on these results and pH-dependent NMR relaxation dispersion measurements, we estimate that under physiological pH (pH 7-8), G·C Hoogsteen base pairs in naked DNA have a population of 0.02-0.002%, as compared to 0.4% for A·T Hoogsteen base pairs, and likely exist primarily as protonated species.

Density Functional Theory Studies on the Oxidation of 5′-dGMP and 5′-dAMP by a Platinum(IV) Complex

Density functional theory has been used to investigate the oxidation of a guanine nucleotide by platinum(IV), a process that can be important in the degradation of DNA. For the first time, we have provided a comprehensive mechanism for all of the steps in this process. A number of intermediates are predicted to occur but with short lifetimes that would make them difficult to observe experimentally. A key step in the mechanism is electron transfer from guanine to platinum(IV), and we show that this is driven by the loss of a chloride ligand from the platinum complex after nucleophilic attack of 5'-phosphate to C8 of guanine. We have investigated several different initial platinum(IV) guanine adducts and shown that the adduct formed from replacement of an axial chlorine ligand in the platinum(IV) complex undergoes oxidation more easily. We have studied adenine versus guanine adducts, and our results show that oxidation of the former is more difficult because of disruption of the aromatic π system that occurs during the process. Finally, our results show that the acidic hydrolysis step to form the final oxidized product occurs readily via an initial protonation of N7 of the guanine.

Conserved phosphoryl transfer mechanisms within kinase families and the role of the C8 proton of ATP in the activation of phosphoryl transfer

BackgroundThe kinome is made up of a large number of functionally diverse enzymes, with the classification indicating very little about the extent of the conserved kinetic mechanisms associated with phosphoryl transfer. It has been demonstrated that C8-H of ATP plays a critical role in the activity of a range of kinase and synthetase enzymes.ResultsA number of conserved mechanisms within the prescribed kinase fold families have been identified directly utilizing the C8-H of ATP in the initiation of phosphoryl transfer. These mechanisms are based on structurally conserved amino acid residues that are within hydrogen bonding distance of a co-crystallized nucleotide. On the basis of these conserved mechanisms, the role of the nucleotide C8-H in initiating the formation of a pentavalent intermediate between the γ-phosphate of the ATP and the substrate nucleophile is defined. All reactions can be clustered into two mechanisms by which the C8-H is induced to be labile via the coordination of a backbone carbonyl to C6-NH2 of the adenyl moiety, namely a "push" mechanism, and a "pull" mechanism, based on the protonation of N7. Associated with the "push" mechanism and "pull" mechanisms are a series of proton transfer cascades, initiated from C8-H, via the tri-phosphate backbone, culminating in the formation of the pentavalent transition state between the γ-phosphate of the ATP and the substrate nucleophile.ConclusionsThe "push" mechanism and a "pull" mechanism are responsible for inducing the C8-H of adenyl moiety to become more labile. These mechanisms and the associated proton transfer cascades achieve the proton transfer via different family-specific conserved sets of amino acids. Each of these mechanisms would allow for the regulation of the rate of formation of the pentavalent intermediate between the ATP and the substrate nucleophile. Phosphoryl transfer within kinases is therefore a specific event mediated and regulated via the coordination of the adenyl moiety of ATP and the C8-H of the adenyl moiety.

Proton Transfers along Hydrogen Bonds in the Tautomerization of Purine

Tautomerization of purine in the water cluster was investigated by the use of DFT calculations. The correlation between the reaction paths and the number of water molecules (n) was examined. For n = 3 and n = 4, concerted reaction paths were obtained. However, for n = 5, a stepwise path including an ion pair intermediate was found with small activation energies. The n = 4 + 3 and n = 4 + 3 + 9 models were calculated to give further small activation energies, where n = 4 constitutes the reaction center and +3 and +3 + 9 denote the number of catalytic water molecules. The combination of the in-plane deprotonation at the N9 site and the out-of-plane protonation at the N7 site makes the n = 4 model probable. Three protonated n = 4 + 3 + 9 routes, a, b, and c, composed of purineH(+)(H(2)O)(4+3+9) were investigated. The n = 4 + 3 moiety is also included in the three routes, and the route c (with the N1 protonation) was found to be most favorable. The purine tautomerization was found to involve the Zundel cation in the ion pair intermediate.

Role of the Pyridine Nitrogen in Pyridoxal 5′-Phosphate Catalysis: Activity of Three Classes of PLP Enzymes Reconstituted with Deazapyridoxal 5′-Phosphate

Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.

A distortion of the aromatic character of 7-azaindole in solid-state after its N7 protonation: linear polarized IR-spectroscopy of embedded chemicals in nematic host

The effect of N7 protonation on the IR-spectroscopic characteristics in solid-state and aromatic character of 7-azaindole (7AI) are elucidated by means of linear-polarized IR-spectroscopy of oriented colloids in nematic host. A comparison between the data of neutral and protonated form of 7AI is done. The experimental data are supported by theoretical ab initio ones and experimental UV-spectroscopic measurements.

PH-Dependent Configurations of a 5-Chlorouracil-Guanine Base Pair

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.