Catalytic reaction mechanism of L-lactate dehydrogenase: an ab initio study

Studies on the catalytic reaction mechanism of L-lactate dehydrogenase have been carried out by using quantum chemical ab initio calculation at HF/6-31G* level. It is found that the interconversion reaction of pyruvate to L-lactate is dominated by the hydride ion H_r transfer, and the transfers of the hydride ionH _r and protonH _r are a quasi-coupled process, in which the energy barrier of the transition state is about 168.37 kJ/mol. It is shown that the reactant complex is 87.61 kJ/mol lower, in energy, than the product complex. The most striking features in our calculated results are that pyridine ring of the model cofactor is a quasi-boat-like configuration in the transited state, which differs from a planar conformation in some previous semiempirical quantum chemical studies. On the other hand, the similarity in the structure and charge between theH _r transfer process and the hydrogen bonding with lower barrier indicates that the H_r transfer process occurs by means of an unusual manner. In addition, in the transition state the electrostatic interaction between the substrate and the active site of LDH is quite strong and the polarization of the carbonyl in the substrate is gradually enhanced accompanying the formation of the transition state. These calculated results are well in accord with the previous experimental studies, and indicate that the charge on the hydride ion H_r is only +0.13e in the transition state, which is in agreement with the reported semiempirical quantum chemical calculations.     

A theoretical study of the mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II

The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods.Molecular dynamics(MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential(GSBP) by CHARMM based on the X-ray crystal structure.Two models of the ternary elongation complex were constructed based on CHARMM MD calculations.All the species including reactants,transition states,intermediates,and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2.Three pathways were explored using the DFT method.The most favorable reaction pathway involves indirect proton migration from the RNA primer 3’-OH to the oxygen atom of-phosphate via a solvent water molecule,proton rotation from the oxygen atom of-phosphate to the-phosphate side,the RNA primer 3’-O nucleophilic attack on the-phosphorus atom,and P-O bond breakage.The corresponding reaction potential profile was obtained.The rate limiting step,with a barrier height of 21.5 kcal/mol,is the RNA primer 3’-O nucleophilic attack,rather than the commonly considered proton transfer process.A high-resolution crystal structure including crystallographic water molecules is required for further studies.     

水催化HBrO与OH自由基反应的微观机理

采用密度泛函理论,分别在B3LYP/6-311++g(d,p)和B3LYP/aug-cc-PVTZ理论水平下,系统研究了无水和水催化的OH自由基与HBrO反应,即HBrO+OH和HBrO+OH+H_2O 2个反应的微观反应机理,给出了所有可能发生的反应路径,并指出能量最低的反应通道.对于没有水参与的反应,由于OH自由基进攻HBrO方式不同,存在顺式方向和反式方向2种进攻方式的反应路径;当有一分子水参与反应时,考虑HBrO H_2O复合物与OH自由基的反应和HBrO与H_2O OH复合物2种反应情况,共发现4条不同的反应路径.这2种反应的所有路径均是在OH自由基提取氢之前以氢键复合物形式存在,反应过程均为无势垒加合过程,总反应为放热反应.水对目标反应起催化作用,有效地降低了反应的势垒,可以加快OH自由基和HBrO的消耗速度.     

两种双核配合物催化PNPP水解反应的机理研究

Cu-Cu(Ⅱ)和Cu-Zn(Ⅱ)草酰胺桥联双核配合物被合成和表征．研究了该同核和异核配合物催化PNPP水解的动力学和机理,建立了一种双核配合物催化PNPP水解的动力学数学模型．结果表明：在缓冲溶液中随着溶液pH的增大,草酰胺桥联双核配合物催化PNPP水解速率提高;配合物中的两个金属离子在催化PNPP水解过程中具有协同效应;这两种草酰胺桥联双核配合物在催化PNPP水解中表现出较好的催化活性．     

3-磷酸甘油醛脱氢酶与酵母线粒体基因组维持蛋白Mgm101的相互作用

采用酵母双杂交方法, 以Mgm101p为诱饵, 筛选酵母cDNA文库. 分离鉴定15个与Mgm101p相互作用的蛋白因子, 其中5个阳性克隆均为GPD1编码的3-磷酸甘油醛脱氢酶(GAPDH). 克隆了GPD1在 S. cerevisiae的同系物ScTDH2基因, 进行绿色荧光蛋白GFP标记、 亚细胞组分分离和蛋白质印迹分析, 结果表明, GAPDH除了在细胞质为糖酵解酶的主要作用外, 可能为多功能蛋白, 在酵母线粒体中与Mgm101p相互作用参与线粒体DNA维持的生物过程.     

Estimation of the biological variation of glucose-6-phosphate dehydrogenase in dried blood spots

Models based on biological variation provide a well-accepted database with reliable information for clinical laboratories for all purposes including screening, diagnosis and follow-up. Newborn screening laboratories use a blood spotted paper matrix to measure the analytes. This matrix medium is certainly different than body fluid matrix medium. After long-term monitoring of the performance of the Glucose-6-Phosphate Dehydrogenase Kit (R&D Diagnostics OSMMR 2000-D G6PD), the results obtained from the variation analysis were statistically evaluated. Analytical coefficient of variation, CV _A, was found to be 5.41%. The CV _A derived from between run assays was 5.32%, while within-subject biological coefficient of variation, CV _I, was 7.26%. Since minimum performance is defined as CV _A< 0.750 CV _I , CV _A should be lower than 5.44%. The analytical bias in calculation of total error was chosen to evaluate the performance of this assay. In this aspect, CV _G, between-subject biological coefficient of variation, was found to be equal to 10.35%. B _A was found to be 4.12%, which is lower than 4.74%, which means that it is acceptable. Therefore, the minimum quality specification for total error allowable is . When the relevant results obtained in this study were substituted in this formula, TE _a was found to be 13.7% for G6PD measurement in dried blood spots on paper filter matrix. It is expected that this figure will be helpful for the performance evaluation of newborn screening laboratories performing G6PD screening. We have been using error grid graphs for the evaluation of our external quality assurance survey results for the last two years, only because there was no data for assays employing filter matrix. Even the TE _a already reported for EDTA whole blood samples used in G6PD assays has been remarkably high, which can easily create the wrong impression that G6PD is not a reliable test to perform from blood spot cards. The present study shows that this assay is adequately reliable even when performed from dried blood spot matrix. However, we believe that the combination of total allowable error, error grid graphs with a well-defined cut off is the best approach to obtain an accurate performance evaluation for this test.Presented at the 10th Conference Quality in the Spotlight, March 2005, Antwerp, Belgium.     

Schiff碱配合物催化过氧化氢氧化苯酚的动力学研究

按照文献方法合成了钴(Ⅱ)和铁(Ⅱ)Schiff碱配合物,研究了它们作为模拟过氧化物酶在缓冲溶液中以及在三种不同的表面活性剂(CTAB,Brij35,LSS)胶束中催化过氧化氢氧化苯酚的反应,结果表明：过氧化氢／配合物物质的量比和胶束微环境对金属配合物催化过氧化氢氧化苯酚的反应有明显的影响;本文所提出的Schiff碱金属配合物催化苯酚氧化反应的机理和动力学数学模型是合理的。     

Oxidation of aminothiols by molecular oxygen catalyzed by copper ions. Stoichiometry of the reaction

Catalysis of oxidation of aminothiols by copper ions was studied depending on the structure of aminothiols and pH of the medium. The catalytic reaction proceeds in the inner coordination sphere of Cu⁺. At pH 7—9, oxidation of bidentate aminothiols involves reduction of O₂ to H₂O₂. At pH 9—13, oxidation of chelating aminothiols is accompanied by reduction of O₂ to H₂O, whereas oxidation of weak-chelating aminothiols still proceeds by the former mechanism. In this process, the thiolate anions coordinated to the Cu⁺ ions lose one electron each and are oxidized to amino disulfides, which go from the inner sphere of the Cu⁺ complex into a solution. Procedures developed for the determination of amino disulfides, the chemiluminescence determination of H₂O₂ in the presence of aminothiols as luminescence quenchers, and a modified polarographic procedure for the determination of O₂ allowed us to establish that oxidation of aminothiols is not accompanied by catalytic decomposition of H₂O₂ that formed.     

钨酸催化氧化环己烯合成己二酸

以钨酸/有机酸性添加剂为催化体系, 在无有机溶剂、相转移剂的情况下, 催化30%过氧化氢氧化环己烯合成己二酸. 当钨酸∶有机酸性添加剂∶环己烯∶过氧化氢＝1∶1∶40∶176(摩尔比, 钨酸用量为2.5 mmol)时, 使用有机酸性添加剂考察钨酸的催化性能, 结果表明以钨酸/间苯二酚催化氧化环己烯的催化效果最优, 反应8 h时己二酸分离产率达90.9%、纯度为～100%; 而不使用有机酸性添加剂时, 己二酸分离产率只有72.1%, 产品纯度为96.2%. 当使用磺酸水杨酸、草酸、水杨酸为有机酸性添加剂时, 随反应时间的增加, 己二酸分离产率均升高, 但反应6 h以后, 己二酸分离产率随时间的变化不明显. 当磺酸水杨酸用量为2.5 mmol时, 己二酸分离产率和纯度均较高. 钨酸-磺酸水杨酸催化体系重复使用五次后, 己二酸分离产率仍可达到80.5%.     

AuCl3催化2-丙炔基-苯胺和丙炔反应机理的研究

采用密度泛函理论方法B3PW91研究了AuCl3催化剂作用下2-丙炔基-苯胺和丙炔环加成生成吲哚衍生物的反应机理。研究结果表明,吲哚衍生物的生成需要经过六个步骤,其中氢迁移过程具有最高的活化自由能为214.22 kJ/mol,是反应的速率控制步骤。整个反应是强放热的,AuCl3催化下反应能够顺利进行,而且AuCl3易于从产物上离去,所以,对于该反应AuCl3是一种有效的催化剂。     

Theoretical study on the mechanism of reaction between 3-hydroxy-3-methyl-2-butanone and malononitrile catalyzed by lithium ethoxide

The The mechanism of reaction between 3-hydroxy-3-methyl-2-butanone and malononitrile for the synthesis of 2-dicyanomethylene-4, 5, 5-trimethyl-2,5-dihydrofuran-3-carbonitrile catalyzed by lithium ethoxide was investigated by density functional theory (DFT). The geometries and the frequencies of reactants, intermediates, transition states and products were calculated at the B3LYP/6-31G(d) level. The vibration analysis and the IRC analysis verified the authenticity of transition states. The reaction processes were confirmed by the changes of charge density at the bond-forming critical point. The results indicated that lithium ethoxide is an effective catalyst in the synthesis of 2-dicyanomethylene-4, 5, 5-trimethyl-2, 5-dihydrofuran-3-carbonitrile from malononi-trile and 3-hydroxy-3-methyl-2-butanone. The activation energy of the reaction with lithium ethoxide was 115.86 kJ·mol⁻¹ less than the uncatalyzed reaction. The mechanism of the lithium ethoxide catalyzed reaction differed from the mechanism of the uncatalyzed reaction.     

Elucidation of the methyl transfer mechanism catalyzed by chalcone O-methyltransferase: a density functional study

The mechanism of the methyl transfer catalyzed by chalcone O-methyltransferase has been computationally investigated by employing the hybrid functional B3LYP. Two models are constructed based on the two conformations of the substrate isoliquiritigenin in the X-ray structure. Our calculations show that the overall reaction is divided into two elementary steps: the water-assisted deprotonation of the substrate by His278 as a catalytic base, followed by the methyl transfer from S-adenosyl-L-methionine (SAM) to the substrate. The calculated rate-limiting barriers for the methyl-transfer step indicate that the catalytic reactions are energetically feasible for both conformations adopted by the substrate.     

Theoretical study of the reaction mechanism of proton transfer in glycinamide

To investigate the tautomerism of glycinamide that is induced by proton transfer, we present detailed theoretical studies on the reaction mechanism of both the isolated gas phase and H₂O‐assisted proton transfer process of glycinamide, using density functional theory calculations by means of the B3LYP hybrid functional. Twenty‐six geometries, including 10 significant transition states, were optimized, and these geometrical parameters are discussed in detail. The relative order of the activation energy for hydrogen atom transfer of all the conformers has been systematically explored in this essay. For the amido hydrogen atom transfer process, the relative order of the activation energy is: IV < II < III < I, while in the carbonic hydrogen atom transfer process, the relative order is IV > II > III > I. Meanwhile, the most favorable structure for both the amido hydrogen atom transfer and the carbonic hydrogen atom transfer has been found. The involvement of the water molecule not only can stabilize the transition states and the ground states, but can also reduce the activation energy greatly. The superior catalytic effect of H₂O has been discussed in detail. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

The effect of poly(ethylene glycol) on the activity and structure of glucose-6-phosphate dehydrogenase in solution

The effect of poly(ethylene glycol), PEG, on the enzymatic activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in the oxidation of glucose-6-phosphate (G-6-P), using NADP+ as co-enzyme was investigated. The enzymatic activity was determined by means of spectrophotometry in three different media: pure Tris–HCl buffer, solution of PEG400 (20 wt.%) and of PEG4000 (20 wt.%), both in buffer. Comparing the enzymatic activity values measured in pure buffer with those in the polymer solutions, an increase in the enzymatic activity of 20% was observed in the presence of PEG400 as well as in PEG4000. Calorimetric studies indicated the absence of preferential interactions between G-6-PDH and PEG400 or PEG4000. Nevertheless, the interaction enthalpy, ΔH_int, between NADP+ and PEG400 and PEG4000 amounted to −9.3 and −26.7 kJ/mol, respectively. Small angle X-ray scattering (SAXS) measurements were performed in a higher concentration range. Data analysis performed from SAXS curves by means of the intra-particle distance distribution function p(r) and Guinier plots yielded for G-6-PDH in pure buffer and PEG400 solutions radius of gyration, R_g, of about 70 Å and in PEG4000 solutions, R_g of about 40 Å. The latter has the same dimension as that found in the dimeric crystallographic structure of G-6-PDH, evidencing that G-6-PDH preserves its dimeric configuration in PEG4000 solution. On the contrary, different aggregates of G-6-PDH are formed in the presence of either buffer or PEG400. These findings show that the presence of PEG in solution can exert an effect on the enzyme structure and activity.     

A simple and efficient large-scale synthesis of 3-hydroxyphthalans via oxa-Pictet-Spengler reaction catalyzed by nanosilica sulfuric acid

Nanosilica sulfuric acid is found to be a new, powerful and reusable heterogeneous catalyst for the rapid synthesis of 3-hydroxyphthalans via condensation of aromatic aldehydes and 3-hydroxybenzyl alcohols under conventional heating and microwave irradiation. Scale-up preparation of these heterocycles is also carried out.     

Bifunctional sulfur-silicon podands as new nucleophilic ionophores in acyl transfer reactions. Influence of monovalent cations on the reaction kinetics

Two bifunctional sulfur-silicon nucleophilic ionophores 3-(trimethoxysilyl)-propanethiol (Nu1) and 3-(tri-2-methoxyethoxysilyl)-propanethiol (Nu2) were used as reagents in the acyl transfer reaction studied by kinetic methods. Nuclear magnetic resonance (NMR) and electro-spray ionisation mass spectrometry (ESI MS) were used for determination of the stoichiometry and stability constants of the complexes made by the podands and lithium or sodium ions in acetonitrile solution.     

CH_2=CHCOOCH_3与O_3反应机理及速率常数的理论研究

采用CBS-QB3方法构建了丙烯酸甲酯(CH_2=CHCOOCH_3)与O_3反应体系的势能剖面并在此基础上利用经典过渡态理论(TST)和Wigner矫正模型计算了标题反应在200K~1200K温度区间内的速率常数kTST/W.研究结果表明,CH_2=CHCOOCH)3与O)3反应首先经过渡态生成一个稳定的五元环中间体,然后按断键位置不同,分别生成产物P1(CH_3OCOCHO+CH_2O_2)和P2(CH)3OCOCHOO+HCHO).此外,速率常数结果显示,在计算温度范围内,标题反应速率常数呈正温度系数效应.294K时,CH_2=CHCOOCH_3与O_3反应速率常数为1.76×10-18cm~3·molecule~(-1)·s~(-1),与所测实验值(0.95±0.07)×10~(-18)cm~3·molecule~(-1)·s~(-1)非常接近.     

Fe3O4 Anisotropic Nanostructures in Hydrogels: Efficient Catalysts for the Rapid Removal of Organic Dyes from Wastewater

Fe₃O₄ anisotropic nanostructures that exhibit excellent catalytic performance are rarely used to catalyze Fenton‐like reactions because of the inevitable drawbacks resulting from traditional preparation methods. In this study, a facile, nontoxic, water‐based approach is developed for directly regulating a series of anisotropic morphologies of Fe₃O₄ nanostructures in a hydrogel matrix. In having the advantages of both the catalytic activity of Fe₃O₄ and the adsorptive capacity of an anionic polymer network, the hybrid nanocomposites have the capability to effect the rapid removal of cationic dyes, such as methylene blue, from water samples. Perhaps more interestingly, hybrid nanocomposites loaded with Fe₃O₄ nanorods exhibit the highest catalytic activity compared to those composed of nanoneedles and nanooctahedra, revealing the important role of nanostructure morphology. By means of scanning electrochemical microscopy, it is revealed that Fe₃O₄ nanorods can efficiently catalyze H₂O₂ decomposition and thus generate more free radicals (^.OH, ^.HO₂) for methylene blue degradation, which might account for their high catalytic activity.