保留型与反转型β-木糖苷酶活性位点的分子动力学模拟

木聚糖是潜在的重要可再生能源, 如何提高其降解效率已成为近年来的研究热点. β-木糖苷酶是木聚糖降解过程中的关键酶之一, 按其水解机制可分为保留型与反转型酶. 目前虽然对于这两种催化机制的研究不断深入, 但很少有工作从溶液环境的角度出发探究它们的差异. 本文采用分子动力学模拟方法, 对4 个典型的β-木糖苷酶进行了显式溶剂模拟研究, 详细分析了酶的催化氨基酸间的距离和质子供体氨基酸pK_a值的动态变化. 结果显示, 反转型酶催化氨基酸间的距离约为0.8-1.0 nm, 大于保留型的0.5-0.6 nm, 与先前对糖苷酶晶体结构的统计分析结果一致. 令人意外的是, 保留型酶的质子供体通过与其附近组氨酸的相互作用, 其pK_a在两个不同的高、低值之间交替变换, 使保留型酶的双取代反应得以发生; 而反转型酶的质子供体则由附近的天冬氨酸调节, 其pK_a稳定在某个较高值, 这可能有利于其在反应pH值下获得水溶液中的氢离子, 进行反转型酶特有的单取代反应. 因此, 本工作加深了人们对β-木糖苷酶保留型与反转型水解机制的认识, 并为后续酶的理性改造与高效利用提供具有指导价值的结构与机理信息.     

The molecular mechanism of photoporphyrin (YHPD)'s photosensitization--laser Raman spectroscopic study of microcosmic and photosensitive damage of YHPD to protein.

Laser Raman spectroscopy was used to investigate the microcosmic and photosensitive damage of YHPD to lysozyme, of which the three-dimensional structure has been elucidated. The experimental results shown by various damages of the main-chain and side-chain of lysozyme are as follows: (i) Phe and Cys are also damaged by photosensitization of YHPD, except for Trp, Tyr, Met, 1/2Cys and His; (ii) the order of the photosensitized sensitivity of various groups of these amino acids have been described; (iii) Trp and Tyr buried in the three-dimensional structure of the protein are damaged very greatly, and (iv) the main-chain conformation of the protein has changed considerably, such as a decrease in orderly structure (alpha-helix, beta-sheet and beta-turn) and a simultaneous increase in random     

Anion influence on extraction and transport of amino acid methyl esters by functionalised calix[4]arene

The liquid–liquid extraction of a series of amino acid methyl esters has been carried out with functionalised calix[4]arene (5,11,17,23-tetrakis(N-methylpiperazino)-25,26,27,28-tetrahydroxycalix[4]arene) from an aqueous phase into a chloroform phase as ion pairs in the presence of picrate ion or tropaeolin 00 as counter ion in order to study the molecular recognition properties of this receptor. The active transport assisted by pH gradient of amino acids as ion pairs through liquid membrane employing the functionalised calix[4]arene as carrier has been investigated. The results showed that the receptor exhibits good extractability towards amino acids and it can also act as carrier through liquid membrane aiming to the separation of amino acids. It was highlighted that the anion nature used as counter ion, the structure of calix[4]arene, and the structure of amino acids are responsible for the experimental results obtained. High yields in both amino acids extraction and transport were obtained for picrate ion used as counter ion.     

Circular Dichroism of Amino Acids: Following the Structural Formation of Phenylalanine

Circular dichroism (CD) is frequently used to assess the secondary structure of peptides and proteins, whereas less attention has been given to their building blocks, that is, single amino acids, as they do not possess a secondary structure. Here, we follow the CD signal of amino acids and reveal that several acids exhibit a unique CD pattern as a function of their concentration. Accordingly, we propose an eight‐level classification of the CD signal of the various amino acids. Special focus is given to the CD pattern of phenylalanine (Phe), for which we observe the formation of an ultra‐narrow CD peak (full width at high maximum of only 5 nm). This CD peak can be attributed to the formation of Phe‐based chiral structural features. Further support for the formation of an ordered structure is given by using NMR, and the additional self‐assembly process of Phe to tubular structures.     

对一种分离测定氨基酸方法的改进

 对Waters公司采用 6 氨基喹啉 N 羟基琥珀酰亚胺基 氨基甲酸酯 (AccQ Tag)柱前衍生化测定氨基酸的方法进行了改进。将流动相流速由原来的 1 0mL/min改变为 2 0mL/min ,用AccQ Tag专用柱 (3 9mmi.d .× 15 0mm ,4μm)在 17 5min(原为 35min) (运行周期为 2 2 5min ,原为 45min)内快速分离测定了 18种氨基酸和牛磺酸。用Nova PakC18柱 (3 9mmi.d .× 15 0mm ,4μm) ,Nova PakC18柱 (4 6mmi.d .× 15 0mm ,4μm) ,SymmetryC18柱 (3 9mmi.d .× 15 0mm ,4μm)和WatersXterraRP 18柱等反相C18柱代替AccQ Tag专用柱 ,均可对氨基酸进行快速分离。     

不同温度下α-氨基酸在CTAB溶液中的体积及折光率的变化

Densities(ρ)and refractive indices(nD)of glycine(Gly),DL-alanine(Ala),DL-valine(Val)(0.02,0.04,0.06,0.08,and 0.10 mol·L-1)in 0.005 and 0.008 mol·L-1 aqueous cetyltrimethylammonium bromide(CTAB)have been measured at 298.15,303.15,308.15,and 313.15 K.The density data have been utilized to calculate apparent molar volumes(φv),partial molar volumes(φ0v),at infinite dilution and partial molar volumes of transfer φ0v(tr)of amino acids.The refractive index data have been used to calculate molar refractivity(RD)of amino acids in aqueous cetyltrimethylammonium bromide.It has been observed that φ0v varies linearly with increasing number of carbon atoms in the alkyl chain of amino acids,and hence,was split to get contributions from the zwitterionic end groups(NH 3,COO-)and methylene group(CH2)of the amino acids.The behaviour of these parameters has been used to investigate the solute-solute,solute-solvent interactions and the effect of cetyltrimethylammonium cation on these interactions.     

Thermodynamic representation of phase equilibrium behavior of aqueous solutions of amino acids by the modified Wilson model

The polymer–electrolyte Wilson model [R. Sadeghi, J. Chem. Thermodyn. 37 (2005) 323–329] which has a molecular thermodynamic framework has been extended to model the vapor–liquid and liquid–solid equilibrium behavior of amino acids and small peptides in aqueous solutions as functions of temperature, ionic strength and amino acid compositions. The utility of the model is demonstrated with a successful representation of the activity coefficients and the solubility of several amino acids in different aqueous solutions and the results are compared with those obtained from the NRTL model.     

Stabilization of zwitterionic structures of amino acids (Gly, Ala, Val, Leu, Ile, Ser and Pro) by ammonium ions in the gas phase

The thermochemistry of gas-phase ion-molecule interactions and structures of a variety of clusters formed between protonated amino acids and either ammonia or amines have been studied by pulsed ionization high-pressure mass spectrometry (HPMS) and ab initio calculations. The enthalpy changes for the association reactions of protonated Gly, Ala, Val, Leu, Ile, Ser, and Pro with ammonia have been measured as -23.2, -21.9, -21.0, -20.8, -20.6, -22.6, and -20.4 kcal mol(-1), respectively. A very good linear relationship exists between the enthalpy changes and the proton affinities (PAs) of the amino acids, with an exception of Ser, where the hydroxyl substituent forms an extra hydrogen bond with ammonia. For the association reaction of protonated proline and methylamine, the measured enthalpy and entropy changes are -26.6 kcal mol(-1) and -30.1 cal mol(-1) K(-1), respectively. The experimental and calculated results indicate that the zwitterionic structure of proline may be well stabilized by CH3NH3(+). For the first time, the interaction strengths between these amino acids and NH4(+) have been obtained, and comparison with Na+ is discussed. Stabilization of zwitterionic structures of a series of amino acids (Gly, Ala, Val, Ser, and Pro) by various ammonium ions (NH4(+), CH3NH3(+), (CH3)2NH2(+), and (CH3)3NH+) has been investigated systematically. Energy decomposition analysis has been performed so that the salt bridge interaction strengths between zwitterionic amino acids and ammonium ions have been obtained. Some generalizations with respect to the relative stability of zwitterionic structures may be drawn. First, as the PA of an amino acid increases, within a series of Gly, Ala, Val, the zwitterionic structure becomes more energetically favorable relative to a non-zwitterionic isomer. Second, as the PA of an amine increases, the zwitterionic structure of a given amino acid within the complex becomes gradually less favorable. Third, compared to the other amino acids, Pro, the only secondary amine among the 20 naturally occurring amino acids, has a much more pronounced tendency to form the zwitterionic structure, which has been confirmed by the experimental results. Finally, substituents on the amino acid backbone that may participate in additional hydrogen bond interactions in non-zwitterionic isomer may render it more stable, as seen in Ser. These organic ammonium ions are found to be able to very effectively stabilize the zwitterionic structure of amino acids, even more effectively than metal ions, which aids significantly in the understanding of why zwitterionic structures exist extensively in biological systems.     

On the use of solid phase ion exchangers for isolation of amino acids from liquid samples and their enantioselective gas chromatographic analysis

This work focuses on the development of a suitable working procedure for preconcentration of amino acids enantiomers from water samples using a solid phase extraction. The three types of ion exchangers with various capabilities have been used. The effect of experimental conditions in SPE procedure employing strong anion exchange (SAX), weak (WCX) and strong cation exchange (SCX) cartridges (such as sample volume, pH, origin of elution solvent and its volume) on effective preconcentration of the model set of amino acids has been studied in detail. The enantiomers of isolated and preconcentrated amino acids have been analysed by GC on three capillary columns coated with chiral selectors. The different amino acids derivatives have been investigated in order to achieve optimal resolution of biogenic amino acids and their enantiomers. The best separation of amino acid enantiomers has been obtained on a Chirasil-L-Val column analysing their N-TFA methyl esters. It has been shown that SCX-SPE cartridge with sulfonic groups attached on silicagel support is most suitable for isolation and preconcentration of amino acids from water samples. For this sample treatment procedure, the overall recovery of extraction process has been calculated as an average value from three measurements. It has been found, that recoveries are practically identical for both enantiomers of a particular amino acid and varies in the range 75-99% depending on the type of amino acid. The effectivity of this sample preparation and GC method has been verified by preconcentration of amino acids from orange juice fortified by racemic mixture of some selected amino acids.     

A comparative study of various computational approaches in calculating the structure of pyridoxal 5'-phosphate (PLP)-dependent beta-lyase protein. The importance of protein environment

Various computational approaches, using molecular mechanics (Amber), semiempirical (AM1), density functional (B3LYP), and various ONIOM methods, have been comparatively investigated for the structure of Escherichia coli NifS CsdB protein. The structure of the entire monomer containing 407 amino acid residues and 579 surrounding water molecules has been optimized. The full geometry optimization in the "active site-only" approach (including only active site atoms) has been found to give the largest root-mean-square (RMS) deviation from the X-ray structure; a much better agreement has been achieved by keeping the atoms leading to the backbones of some amino acids frozen in their positions in the X-ray structure. The best agreement has been attained by including the surrounding protein in the calculations using the two-layer ONIOM (B3LYP:Amber) approach. The results presented in this study conclusively demonstrate the importance of the protein/active-site interaction on the active-site structure of the enzyme. The present theoretical study represents the largest system studied at the ONIOM level to date, containing 7992 atoms, including 84 atoms in the QM region and rest in the MM region.     

CE methods for the determination of non-protein amino acids in foods

In addition to the 20 amino acids universally distributed as protein constituents in living organisms, there are other amino acids of non-protein origin that can be found in foods. The determination of these non-protein amino acids is interesting since they can be indicative of the quality and safety of foods. This work presents for the first time an updated and comprehensive review devoted to show the possibilities of capillary electrophoresis for the determination of non-protein amino acids in food samples. The results reported have been classified according to the chemical structure of the non-protein amino acid studied. Separation conditions as well as detection systems used have been detailed since most of these amino acidic compounds do not possess chromophore groups detectable by conventional UV-Vis detection, being in this case necessary a previous derivatization step. Finally, the application of microchip electrophoresis to the determination of non-protein amino acids in foodstuffs is also included in this review.     

Synthesis and application of dehydroabietylisothiocyante as a new chiral derivatizing agent for the enantiomeric separation of chiral compounds by capillary electrophoretic

A new chiral derivatizing reagent, dehydroabietylisothiocyante (DHAIC), was synthesized and used for the enantiomeric separation of chiral compounds in capillary electrophoresis (CE). The synthetic route to obtain DHAIC is described. The separation conditions for the chiral separation of several chiral compounds, such as protein amino acids and chiral drug DOPA were optimized. Best results for the chiral separation of DHAIC derivatized amino acids and DOPA were obtained in a running buffer consisted of 50 mM borate (pH 9.5), 5 mM sodium dodecyl sulphate (SDS) and 20% acetonitrile for amino acids and 60 mM Na₂HPO₄ (pH 8.0), 17 mM SDS and 25% acetonitrile for DOPA. Under the conditions studied, chiral separation of five amino acids including Ser, Val, Ala, Thr, Cys and a chiral drug DOPA as their diastereomeric DHAIC derivatives has been achieved by micellar electrokinetic chromatography (MEKC).     

X-ray K-absorption spectra of some aminoacid and carbonato complexes of copper(II)

Summary X-ray (K-) absorption edge spectrometric (XAES) investigations of some copper(II) chelates involving amino acids and a carbonato complex have been carried out employing a curved crystal spectrograph. Experimental edgewidths have been used to bear on the number of nearest neighbours around the central metal atom in the complexes in the solid state. Comparisons in respect of the nature of the metal-ligand bond have been made between inferences drawn on the basis of XAES, i.r. and optical spectral data. Average radii of the coordination sphere in the amino acid chelates have been reported using extended fine structure energy separations (Levy's method). In the carbonato complex, the possibility of determining different metal-donor distances within the coordination sphere has been suggested for the first time.     

Uncharged water-soluble Co(II)-porphyrin: a receptor for aromatic alpha-amino acids

Changes in the UV-vis spectra and induced circular dichroism (ICD) signals observed, in correspondence with the porphyrin Soret region, for aqueous solutions of achiral 5,10,15,20-tetrakis{p-[omega-methoxy poly(oxy-ethylene)]phenyl}porphyrin cobalt (II) (Co-P) and aromatic alpha-L-amino acids (Trp and Phe) give direct evidence for the coordination between the Co-P and amino acids. Considering that Co-P, besides the Co atom (one-fixation-point system), does not contain in the molecule active ligand groups and that no ICD signals have been observed in the case of Co-P/Ala, it has been concluded that hydrophobic interactions or stacking interactions between the aromatic rings of the porphyrin and those of Trp or Phe, acting as further amino acid (AA) fixation points, can strongly reduce the mobility of the chiral guest, thus permitting the generation of ICD signals. The effects of changes of both pH (in the range 2-9) and amino acid structure on the ICD phenomenon have also been investigated. In particular, the following have been observed: (i) strong ICD signals for all of the Co-P/N-acetyl amino acid aqueous solutions at pH 7, (ii) an unexpected ICD band with a bisignate form for the Co-P/Ala solution at pH 9 after long aging, and (iii) an opposite ICD signal when alpha-D-Phe and alpha-D-Trp enantiomers have been used. The data reported in this paper show how the binding mechanism between receptor and AAs changes by modulating properly the pH or the molecular structures and indicate that in these aqueous solutions the coordination Co-N is not the fundamental mechanism giving rise to the formation of the complexes and that the binding can be driven by hydrophobic interactions. These occurrences, through the analysis of the spectroscopic response (and, in particular, the form of the ICD band), can allow the recognition of AAs.     

New triazine spectroscopic reagent for the separation of DL-amino acids by micellar electrokinetic chromatography

An approach to the chiral separation of racemic mixtures of amino acids by means of micellar electrokinetic chromatography after derivatization with a new triazine spectroscopic reagent, 3-(4,6-dichloro-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine (DTDP), has been evaluated. It was found that the derivatives of the aliphatic amino acids such as serine, valine and arginine, could produce a strong UV absorption at 282 nm, whose apparent molar absorptivities are of 10(-4) M(-1) cm(-1), and thus the concentration of the amino acids down to 3 x 10(-7) M can still give a detectable signal (S/N = 3). Beta-Cyclodextrin (beta-CD) added to the buffer system was used as a chiral selector, and separation conditions were optimized. The presence of an organic modifier (2-propanol) was also a prerequisite for the chiral separation. The best results for the chiral separation of DTDP-amino acids were achieved in a mixed sodium dodecylsulfate-beta-CD-borate-2-propanol medium at pH 9.0. Compared to some of the commonly used derivatization methods, the present one offers a relatively stable derivative and strong UV absorption for the spectroscopically inert amino acids, thus enabling amino acids to be separated and detected by CE even with a simpler UV detector.     

Koshikamide B, a cytotoxic peptide lactone from a marine sponge Theonella sp

Koshikamide B (1) has been isolated from two separate collections of the marine sponge Theonella sp. as the major cytotoxic constituent. Koshikamide B is a 17-residue peptide lactone composed of six proteinogenic amino acids, two D-isomers of proteinogenic amino acids, seven N-methylated amino acids, and two unusual amino acid residues. The unusual amino acids are N(delta)-carbamoylasparagine and 2-(3-amino-2-hydroxy-5-oxopyrrolidin-2-yl)propionic acid (AHPP); the former is first found as the constituent of peptides, whereas the latter is a new amino acid residue. The N-terminus of koshikamide B is blocked by a methoxyacetyl group. The structure of koshikamide B (1) has been determined by interpretation of spectral data and analysis of chemical degradation products. Koshikamide B (1) exhibits cytotoxicity against P388 murine leukemia cells and the human colon tumor (HCT-116) cell line with an IC50 value of 0.45 and 7.5 microg/mL, respectively.     

One-pot, large-scale synthesis of Nickel(II) complexes derived from 2-[N-(alpha-picolyl)amino]benzophenone (PABP) and alpha- or beta-amino acids

A one-pot, large-scale procedure for preparing the Belokon-Soloshonok nucleophilic glycine equivalent 2-[N-(alpha-picolyl)amino]benzophenone (PABP) derived Ni(II) complex [GlyNi(II)PABP] is described. It has been accomplished by using isobutyl chloroformate to form PABP and then NaH/KOH as mixed bases to afford the corresponding complexes in a one-pot manner (up to an overall yield of 98%). The potential of this method for preparation of beta-amino acids derivatives, such as beta-AlaNi(II)PABP and beta-PheNi(II)PABP, has been demonstrated. The structure of beta-AlaNi(II)PABP is characterized by single-crystal X-ray diffraction.     

Spectral representation of reduced protein models

We consider a spectrum-like two-dimensional graphical representation of proteins based on a reduced protein model in which 20 amino acids are grouped into five classes. This particular grouping of amino acids was suggested by Riddle and co-workers in 1997. The graphical representation is based on depicting sequentially the amino acids on five horizontal lines at equal separations. One-letter codes, B, O, U, X and Y, to which numerical values 1 to 5 have been assigned, are suggested as labels for the fictional amino acids that represent all the amino acids within each group. The approach is illustrated on ND6 proteins of eight species having from 168 to 175 amino acids. While visual inspection of the novel spectral graphical representations of proteins may reveal local similarities and dissimilarities of protein sequences, arithmetic manipulations of spectra offer an elegant route to graphic visualization of the degree of similarity for selected pairs of proteins.