A theoretical study of the<Emphasis Type="Italic"> cis</Emphasis>-dihydroxylation mechanism in naphthalene 1,2-dioxygenase

The catalytic mechanism of naphthalene 1,2-dioxygenase has been investigated by means of hybrid density functional theory. This Rieske-type enzyme, which contains an active site hosting a mononuclear non-heme iron(II) complex, uses dioxygen and two electrons provided by NADH to carry out the cis-dihydroxylation of naphthalene. Since a (hydro)peroxo-iron(III) moiety has been proposed to be involved in the catalytic cycle, it was probed whether and how this species is capable of cis-dihydroxylation of the aromatic substrate. Different oxidation and protonation states of the Fe–O₂ complex were studied on the basis of the crystal structure of the enzyme with oxygen bound side-on to iron. It was found that feasible reaction pathways require a protonated peroxo ligand, Fe^III–OOH; the deprotonated species, the peroxo-iron(III) complex, was found to be inert toward naphthalene. Among the different chemical patterns which have been explored, the most accessible one involves an epoxide intermediate, which may subsequently evolve toward an arene cation, and finally to the cis-diol. The possibility that an iron(V)-oxo species is formed prior to substrate hydroxylation was also examined, but found to implicate a rather high energy barrier. In contrast, a reasonably low barrier might lead to a high-valent iron-oxo species [i.e. iron(IV)-oxo] if a second external electron is supplied to the mononuclear iron center before dioxygenation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0537-0     

Modeling of the mechanism of nucleophilic aromatic substitution of fungicide chlorothalonil by glutathione

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TCIN, CAS 1897-45-6) is a broad range spectrum fungicide whose fungitoxic action has been associated with the rapid formation of conjugated chlorothalonil–cellular thiol derivatives, specifically with thiol-rich enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and with glutathione (GSH). The biotransformation reaction sequence between enzyme-activated glutathione (GSH) and chlorothalonil depletes cellular glutathione reserves. The conjugation of glutathione with chlorothalonil via nucleophilic aromatic substitution was modeled for an isolated reacting species using semiempirical self-consistent field molecular orbital (SCF-MO) theory at the PM3 level. The potential energy hypersurface at each of the three possible chlorinated attack sites on chlorothalonil was elaborated using a thiolate (CH₃S^–) anion as a model for an enzyme-activated glutathione molecule. Calculated free energies of activation for formation of mono-RSH conjugates suggest that the order of nucleophilic attack on chlorine positions in TCIN is 2>4, 6>5 although energy differences are small (on the order of 1–2 kcal mol^–1). Meisenheimer or -complexes have been isolated as true intermediates on the hypersurface for each reaction, suggesting that the mechanism follows a two-step pathway.Electronic Supplementary Material available.     

Synthesis of novel 3-pyridinecarbonitriles with amino acid function and their fluorescence properties

Summary. A variety of N-[(4,6-diaryl-3-pyridinecarbonitrile)-2-yl] amino acid esters 2–4 were synthesized through the reaction of 2-bromo-3-pyridinecarbonitriles 1 with the appropriate -amino acid ester hydrochloride in refluxing dioxane in the presence of triethylamine as dehydrohalogenating agent. Similarly, N-glycylglycine analogues 5 were obtained through the reaction of 1 with the dipeptide ester. On the other hand, attempts were made towards the construction of amino acid derivatives 7 through the reaction of 1 with aqueous solution -amino acids 6 in refluxing pyridine, but were unsuccessful, and instead the unexpected 2-amino-3-pyridinecarbonitriles 8 were isolated. The fluorescence properties of the newly synthesized pyridines 2–5 were evaluated. Some of the prepared compounds show considerable antibacterial activity.     

Theoretical studies of biliverdin: energetics of the reduction pathways to bilirubin

Geometries and energies of formation of bilirubin formed by reduction of biliverdin via three meso carbon sites, the , and positions, have been calculated using semiempirical methods. It has been shown that -bilirubin with a ridge-tile conformation forms six intramolecular hydrogen bonds and is the most stable of the three above mentioned positions by at least 22 kcal mol^–1. Reduction pathways for -, - and -bilirubin formations from biliverdin are studied in detail. The roles of loss of conjugation and hydrogen bond formations in stability of different conformers have been discussed. -Bilirubin was fully optimized by using ab initio methods. Fine refinements of calculated results show excellent agreement with experimental results. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0078-9.Electronic Supplementary Material available.     

Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate‐limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol^?1 compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol^?1, in excellent agreement with the experimental value of 16.5 kcal mol^?1. Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction. Proteins 2014; 82:3347–3355. © 2014 Wiley Periodicals, Inc.     

Homology modeling reveals the structural background of the striking difference in thermal stability between two related [NiFe]hydrogenases

Hydrogenases are redox metalloenzymes in bacteria that catalyze the uptake or production of molecular hydrogen. Two homologous nickel–iron hydrogenases, HupSL and HydSL from the photosynthetic purple sulfur bacterium Thiocapsa roseopersicina, differ substantially in their thermal stabilities despite the high sequence similarity between them. The optimum temperature of HydSL activity is estimated to be at least 50 °C higher than that of HupSL. In this work, homology models of both proteins were constructed and analyzed for a number of structural properties. The comparison of the models reveals that the higher stability of HydSL can be attributed to increased inter-subunit electrostatic interactions: the homology models reliably predict that HydSL contains at least five more inter-subunit ion pairs than HupSL. The subunit interface of HydSL is more polar than that of HupSL, and it contains a few extra inter-subunit hydrogen bonds. A more optimized cavity system and amino acid replacements resulting in increased conformational rigidity may also contribute to the higher stability of HydSL. The results are in accord with the general observation that with increasing temperature, the role of electrostatic interactions in protein stability increases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00894-001-0071-8.Electronic Supplementary Material available.     

The effect of sugar,amino acid,metal ion,and NaCl on model Maillard reaction under pH control

Summary. The color intensities was determined of Maillard reaction products (MRPs) prepared by heating each of five sugars (maltose, fructose, glucose, arabinose, and xylose) with each of 12 amino acids (aspartic acid, glutamic acid, alanine, leucine, isoleucine, valine, proline, serine, cysteine, phenylalanine, arginine, and lysine). The remaining percentages of glucose and rate of change of color intensity due to the addition of a metal ion and NaCl were monitored for nine MRPs that had been formed between glucose and each of nine amino acids (aspartic acid, glutamic acid, alanine, valine, serine, cysteine, phenylalanine, arginine, and lysine). Model MRPs were prepared in a block heater at 100°C for 1–12h with the pH value controlled at 6.5. The resulting color intensity of each MRPs formed from the basic amino acids was greater due to the higher reactivity than those from the acidic amino acids. The remaining percentage of glucose in each MRPs from the basic amino acids was lower than those from the acidic amino acids. The MRPs from the nonpolar amino acids showed an intermediate color intensity and remaining percentages of glucose between those formed from the basic and acidic amino acids. Browning tended to be accelerated in the presence of metal ions, especially Fe²⁺ and Cu²⁺, although it was affected by the property of the amino acid and heating time as well as by the type of metal ion. On the other hand, browning was greatly inhibited by a high concentration of NaCl.     

Marfey’s reagent for chiral amino acid analysis: A review

Summary. The present paper describes characteristics and application of Marfeys reagent (MR) including general protocols for synthesis of the reagent and diastereomers along with advantages, disadvantages and the required precautions. Applications, and comparison with other derivatizing agents, for the resolution of complex mixtures of DL-amino acids, amines and non-proteinogenic amino acids, peptides/amino acids from microorganisms, cysteine residues in peptides, and evaluation of racemizing characteristics have been discussed. Separation mechanisms of resolution of amino acid diastereomers and replacement of Ala–NH₂ by suitable chiral moieties providing structural analogs and different chiral variants and their application as a derivatizing agent to examine the efficiency, and reactivity of the reagent have been focussed. Use of MR for preparing CSPs for direct enantiomeric resolution has also been included.On leave from Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247 667, India.     

Structural basis for substrate specificity of l‐methionine decarboxylase

l ‐Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B₆‐dependent enzyme and catalyzes the non‐oxidative decarboxylation of l ‐methionine to produce 3‐methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand‐free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.     

Hydropathic analysis of the free energy differences in anthracycline antibiotic binding to DNA

Molecular models of six anthracycline antibiotics and their complexes with 32 distinct DNA octamer sequences were created and analyzed using HINT (Hydropathic INTeractions) to describe binding. The averaged binding scores were then used to calculate the free energies of binding for comparison with experimentally determined values. In parsing our results based on specific functional groups of doxorubicin, our calculations predict a free energy contribution of –3.6 ± 1.1 kcal mol^–1 (experimental –2.5 ± 0.5 kcal mol^–1) from the groove binding daunosamine sugar. The net energetic contribution of removing the hydroxyl at position C9 is –0.7 ± 0.7 kcal mol^–1 (–1.1 ± 0.5 kcal mol^–1). The energetic contribution of the 3′ amino group in the daunosamine sugar (when replaced with a hydroxyl group) is –3.7 ± 1.1 kcal mol^–1 (–0.7 ± 0.5 kcal mol^–1). We propose that this large discrepancy may be due to uncertainty in the exact protonation state of the amine. The energetic contribution of the hydroxyl group at C14 is +0.4 ± 0.6 kcal mol^–1 (–0.9 ± 0.5 kcal mol^–1), largely due to unfavorable hydrophobic interactions between the hydroxyl oxygen and the methylene groups of the phosphate backbone of the DNA. Also, there appears to be considerable conformational uncertainty in this region. This computational procedure calibrates our methodology for future analyses where experimental data are unavailable.     

Tryptophanase in aqueous methanol: the solvent effects and a probable mechanism of the hydrophobic control of substrate specificity

To shed light on the mechanism of hydrophobic control in reactions of microbial tryptophanase the direct effect of the solvent hydrophobicity on affinities of amino acid inhibitors was first examined. Values of inhibition constants (K_i) for a variety of amino acids were determined in 37.5% aqueous methanol, and no general correlation between the change of K_i, on passing from water to aqueous methanol, and amino acid hydrophobicity was found. The solvent effects on the separate stages of the external aldimine formation (K_D) and deprotonation to form a quinonoid intermediate (K_q) were determined for the reactions of tryptophanase with 2-oxindolyl- -alanine and -alanine by stopped-flow technique. For 2-oxindolyl- -alanine, which is a close transition-state analogue for the enzyme reaction with natural substrate, the decrease in the affinity in aqueous methanol is associated exclusively with the α-proton abstraction stage but not with the preceding formation of external aldimine. We conclude that the environment of amino acid side chains in the active site cannot be considered to be permanently hydrophobic irrespective of the bound amino acid. We suggest that complexes of tryptophanase with amino acids may exist either in a hydrophobic, presumably “closed”, conformation, where bound amino acids are isolated from the solvent, or in an accesible to solvent, “open”, conformation, depending on the structure of the bound amino acid and stage of the catalytic mechanism. For 2-oxindolyl- -alanine the transfer from an open to a closed conformation probably accompanies deprotonation of the external aldimine. The change of the active site hydrophobicity may provide an efficient way of modulating the relative acid–base properties of the catalytic groups to ensure the movement of protons in the “correct” direction depending on the elementary stage of catalysis.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Proteolytic specificity of chicken cathepsin L on bovineβ-casein

The proteolytic specificity of chicken cathepsin L was studied using bovine -casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P ₁ . Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P ₁ –P ₂ positions and that the S ₁ subsite can interact with modified amino acids such as phosphoserine.Abbreviations RP-HPLC reverse phase high performance liquid chromatography - NMec N-methyl coumarylamide - TEA triethylamine - TFA trifluoroacetic acid     

Mediated exodus of L-dopa from human epidermal Langerhans cells

Summary. L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, -methyl-dopa (-m-dopa), D-dopa and the dipeptide, met–ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5–7min and a complete exodus of L-dopa was attained at about 20min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of -m-dopa, D-dopa and met–ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.     

On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

Acid-base chemistry of vitamin B6 compounds in methanol

The enzymatic reactions involving pyridoxal 5-phosphate (PLP) can be simulated in solutions; thus, this system forms a favorable model for understanding the requirements of the enzymatic catalysis. We have studied in methanol protonic equilibria of the imines formed between PLP or salicylaldehyde (SA) and various amino acids, using UV and NMR spectroscopy. A glass electrode and an operationalpH* scale were used to control acidity. The first protonation of the phosphate of PLP imines can be detected by UV spectroscopy withpK* at 10.8, proved by [³¹P]-NMR. The second protonation of phosphate (pK* at 4.8) is accompanied by increased hydrolysis of the imines. The imines of aspartate deviate from the imines of nondicarboxylic amino acids indicating that the -carboxyl of aspartate is internally hydrogen-bonded. PLP-2-aminobutanol Schiff base does not show with [¹H]-NMR atpH* 7 separate peaks for ketoenamine-enolimine tautomers even at -90°C, SA-phenylalanine shows an unidentified absorption at 350–380 nm. This was tentatively assigned a trans structure.     

Peroxynitrite reactivity with amino acids and proteins

Summary. Peroxynitrite, the product of the fast reaction between nitric oxide (NO) and superoxide O₂ ^– radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Responsiveness of cardiac Na+ channels to a site-directed antiserum against the cytosolic linker between domains III and IV and their sensitivity to other modifying agents

Elementary Na⁺ currents were recorded in inside-out patches from neonatal rat heart cardiocytes to analyze the influence of a site-directed polyclonal anti-serum against the linker region between the domains III and IV (amino acids 1489–1507 of the cardiac Na⁺ channel protein) on Na⁺ channel gating and to test whether this part of the -subunit may be considered as a target for modifying agents such as the (–)-enantiomer of DPI 201-106.Anti-SLP 1 serum (directed against amino acids 1490–1507) evoked, usually within 10–15 min after cytosolic administration, modified Na⁺ channel activity. Antiserum-modified Na⁺ channels retain a single open state but leave, at –60 mV for example, their conducting configuration consistently with an about threefold lower rate than normal Na⁺ channels. Another outstanding property of noninactivating Na⁺ channels, enhanced burst activity, may be quite individually pronounced, a surprising result which is difficult to interpret in terms of structure function relations. Removal of inactivation led to an increase of reconstructed peak I _Na (indicating a rise in NP _o) and changed I _Na decay to obey second-order kinetics, i.e., open probability declined slowly but progressively during membrane depolarization. The underlying deactivation process is voltage dependent and responds to a positive voltage shift with a deceleration but may operate even at the same membrane potential with different rates. Iodatemodified Na⁺ channels exhibit very similar properties including a conserved conductance. They are likewise controlled by an efficient, voltage-dependent deactivation process. Modification by (–)-DPI 201-106 fundamentally contrasts to the influence of anti-SLP 1 serum and the protein reagent iodate since (–)-DPI-modified Na⁺ channels maintain their open probability for at least 120 msec, i.e., a deactivation process seems lacking. This functional difference suggests that the linker region between the domains III and IV of the -subunit may not be the only target for (–)-DPI 201-106 and related compounds, if at all.This work was supported by a grant of the Deutsche Forschungs-gemeinschaft (Ko 778/2–4), Bonn.