Exploring the active-site of a rationally redesigned lipase for catalysis of Michael-type additions

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant lacks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The kca values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.     

Fluoride catalysis at 175°C. of the reaction: Glycerol plus fatty acid

1. In uncatalyzed reactions the esterification of stearic, oleic, and linolenic acid proceeded at the same general rate though the speed was somewhat enhanced as the degree of unsaturation of the fatty acid increased. None of the reactions went to completion, after 15 hours being 72%, 83%, and 84%, respectively, completed. Only a small fraction of the distearate was converted to the triglyceride; a greater amount of the dioleate was esterified to the trioleate and about half the dilinolenate was changed to the trilinolenate.
2. Of 15 metallic fluorides studied, all afforded some catalysis, but only antimony trifluoride and zinc and cobalt fluorides were outstanding. Antimony trifluoride was the most active although in large amount it caused severe decomposition and charring of the products. Zinc fluoride had the best general properties, approaching antimony trifluoride yet causing practically no decomposition even in high concentration. Its activity was enhanced with an increase in amount, the best concentration apparently being 0.1 mole per 100 grams fatty acid. Antimony trifluoride was also better in larger amount within limits, but cobalt fluoride did not exhibit this property.
3. With zinc fluoride as catalyst the reactions of stearic, oleic and linolenic acids after 15 hours were 95%, 94%, and 92%, respectively, completed. At least three-fourths of the diglycerides were converted to triglycerides. From a study of reaction velocities, zinc fluoride caused the monoglyceride to form at a rate approximately four times that of the uncatalyzed reaction and the diglyceride at a six-fold rate. The triglyceride was formed at an almost undiminished rate whereas in the uncatalyzed reaction the formation of triglyceride was diminished rapidly.
4. Unsaturation and viscosity were not affected seriously in the presence of the fluorides except antimony trifluoride. Certain other fluorides caused severe darkening but without accompanying serious decomposition.

     

Redox tuning over almost 1 V in a structurally conserved active site: lessons from Fe-containing superoxide dismutase

Metalloenzymes catalyze some of the most demanding reactions in biochemistry, thereby enabling organisms to extract energy from redox reactions and utilize inorganic starting materials such as N 2 and CH 4. Bound metal ions bring to enzymes greater chemical versatility and reactivity than would be possible from amino acids alone. However the host proteins must control this broad reactivity, activating the metal for the intended reaction while excluding the rest of its chemical repertoire. To this end, metalloproteins must control the metal ion reduction midpoint potential ( E m), because the E m determines what redox reactions are possible. We have documented potent redox tuning in Fe- and Mn-containing superoxide dismutases (FeSODs and MnSODs), and manipulated it to generate FeSOD variants with E ms spanning 900 mV (21 kcal/mol or 87 kJ/mol) with retention of overall structure. This achievement demonstrates possibilities and strategies with great promise for efforts to design or modify catalytic metal sites. FeSODs and MnSODs oxidize and reduce superoxide in alternating reactions that are coupled to proton transfer, wherein the metal site is believed to cycle between M3+ x OH- and M2+ x OH2 (M = Fe or Mn). Thus the E m reflects the ease both of reducing the metal ion and of protonating the coordinated solvent molecule. Moreover similar E ms are achieved by Fe-specific and Mn-specific SODs despite the very different intrinsic E(m)s of high-spin Fe3+/2+ and Mn3+/2+. We provide evidence that E(m) depression by some 300 mV can be achieved via a key enforced H-bond that appears able to disfavor proton acquisition by coordinated solvent. Based on 15N-nuclear magnetic resonance (NMR), stronger H-bond donation to coordinated solvent can explain the greater redox depression achieved by the Mn-specific SOD protein compared with the Fe-specific protein. Furthermore, by manipulating the strength and polarity of this one H-bond, with comparatively minor perturbation to active site atomic and electronic structure, we succeeded in raising the E m of FeSOD by more than 660 mV, apparently by a combination of promoting protonation of coordinated solvent and providing an energetically favorable source of a redox-coupled proton. These studies have combined the use of electron paramagnetic resonance (EPR), NMR, magnetic circular dichroism (MCD), and optical spectrophotometry to characterize the electronic structures of the various metal sites, with complementary density functional theoretical (DFT) calculations, NMR spectroscopy, and X-ray crystallography to define the protein structures and protonation states. Overall, we have generated structurally homologous Fe sites that span some 900 mV, and have demonstrated the enormous redox tuning accessible via the energies associated with proton transfer coupled to electron transfer. In this regard, we note the possible significance of coordinated solvent molecules in numerous biological redox-active metal sites besides that of SOD.     

Exploring the conserved water site and hydration of a coiled-coil trimerisation motif: a MD simulation study

The solvent structure and dynamics around ccbeta-p, a 17-residue peptide that forms a parallel three-stranded alpha-helical coiled coil in solution, was analysed through 10 ns explicit solvent molecular dynamics (MD) simulations at 278 and 330 K. Comparison with two corresponding simulations of the monomeric form of ccbeta-p was used to investigate the changes of hydration upon coiled-coil formation. Pronounced peaks in the solvent density distribution between residues Arg8 and Glu13 of neighbouring helices show the presence of water bridges between the helices of the ccbeta-p trimer; this is in agreement with the water sites observed in X-ray crystallography experiments. Interestingly, this water site is structurally conserved in many three-stranded coiled coils and, together with the Arg and Glu residues, forms part of a motif that determines three-stranded coiled-coil formation. Our findings show that little direct correlation exists between the solvent density distribution and the temporal ordering of water around the trimeric coiled coil. The MD-calculated effective residence times of up to 40 ps show rapid exchange of surface water molecules with the bulk phase, and indicate that the solvent distribution around biomolecules requires interpretation in terms of continuous density distributions rather than in terms of discrete molecules of water. Together, our study contributes to understanding the principles of three-stranded coiled-coil formation.     

Synergy at a distance in the synthesis of methanol over copper catalysts

Physical mixtures of Cu/SiO₂ and ZnO/SiO₂ catalysts have been studied for the synthesis of methanol from CO/CO₂/H₂ at 250 °C and 10 bar pressure. It is found that the activities are very much higher for the physical mixtures than would be expected from the activities of either of these two catalysts in isolation. The results suggest that the high activity of conventional Cu/ZnO/Al₂O₃ catalysts may arise from a synergy between the Cu and ZnO phases.     

N-末端核定位序列对猪圆环病毒衣壳蛋白病毒样颗粒在汉逊酵母中组装的影响

目的探讨N-末端核定位序列(nuclear localization signal,NLS)对猪圆环病毒2型(porcine circovirus type 2,PCV2)衣壳蛋白病毒样颗粒(virus-like particle,VLP)在汉逊酵母(Hansenula polymorpha,HP)中组装的影响。方法分别构建表达PCV2 ORF2全长及NLS截短型重组表达质粒p MOXZ-full和p MOXZ-ΔN40,电转至HP,构建重组酵母表达菌株full-HP和ΔN40-HP,获得的两种表达产物经蔗糖密度梯度超速离心及超滤后,Western blot法检测两种表达产物的单体蛋白表达和VLP组装效率,透射电子显微镜观察VLP形态,小鼠免疫试验检测VLP的免疫原性,地高辛标记DNA杂交法检测VLP的DNA结合能力。结果与全长ORF2表达产物PCV2-VLP(full)比较,NLS截短型表达产物PCV2-VLP(ΔN40)的单体表达量略有提高,但VLP组装效率及DNA结合能力均降低。两种VLP颗粒形态相近,直径约20 nm;两种VLP免疫小鼠血清的抗体几何平均滴度(GMT)差异无统计学意义(P0.05)。结论去除NLS可提高PCV2衣壳蛋白VLP单体的表达水平,但不是VLP组装所必须的,且可使VLP的组装率略有降低。本研究为PCV2-VLP结构和疫苗的研究奠定了基础。     

Electrostatic stabilization and general base catalysis in the active site of the human protein disulfide isomerase a domain monitored by hydrogen exchange

The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the alpha(2) helix. Amides in the active site exhibit diffusion-limited, hydroxide-catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide-catalyzed exchange by a million-fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition-state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.     

Perception byTrogoderma species of chirality and methyl branching at a site far removed from a functional group in a pheromone component

Responses to enantiomers of (Z)- and (E)-trogodermal (14-methyl-8-hexadecenal) suggest that fourTrogoderma species utilize the (R)-(–) configuration at C-14. Removal of the C-14 methyl branch decreased the response. These results demonstrate the high specificity associated with the configuration at a chiral center, or the methyl branch, distant in terms of numbers of bonds from a functional group.Coleoptera: Dermestidae.     

Substrate binding and catalysis in trichosanthin occur in different sites as revealed by the complex structures of several E85 mutants

Trichosanthin (TCS) is a type I ribosome-inactivating protein(RIP) which possesses rRNA N-glycosidase activity. In recentyears, its immunomodulatory, anti-tumor and anti-HIV propertieshave been revealed. Here we report the crystal structures ofseveral E85 mutant TCS complexes with adenosine-5'-monophosphate(AMP) and adenine. In E85Q TCS/AMP and E85A TCS/AMP, near theactive site of the molecule and parallel to the aromatic ringof Tyr70, an AMP molecule is bound to the mutant without beinghydrolyzed. In the E85R TCS/adenine complex, the hydrolyzedproduct adenine is located in the active pocket where it occupiesa position similar to that in the TCS/NADPH complex. Significantly,AMP is bound in a position different to that of adenine. Incomparison with these structures, we suggest that there areat least two subsites in the active site of TCS, one for initialsubstrate recognition as revealed by the AMP site and anotherfor catalysis as represented by the NADPH site. Based on thesecomplex structures, the function of residue 85 and the mechanismof catalysis are proposed. Received December 10, 2002; accepted May 20, 2003.     

浅谈建筑施工场界噪声标准存在的问题与修订要点

在提出目前国家《建筑施工场界噪声限值》和《建筑施工场界噪声测量方法》标准中存在一些问题的同时,阐述该标准修订的必要性、紧迫性与修订要点.     

Change in the crystal packing of soybean {beta}-amylase mutants substituted at a few surface amino acid residues

In spite of the high similarity of amino acid sequence and three-dimensionalstructure between soybean ß-amylase (SBA) and sweetpotato ß-amylase (SPB), their quaternary structureis quite different, being a monomer in SBA and a tetramer inSPB. Because most of the differences in amino acid sequencesare found in the surface region, we tested the tetramerizationof SBA by examining mutations of residues located at the surface.We designed the SBA tetramer using the SPB tetramer structureas a model and calculating the change of accessible surfacearea (ASA) for each residue in order to select sites for themutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D)and SB4 (K462S added to SB3), were constructed for expressionin Escherichia coli and the recombinant proteins were purified.They existed as a monomer in solution, but gave completely differentcrystals from the native SBA. The asymmetric unit of the mutantscontains four molecules, while that of native SBA contains one.The interactions of the created interfaces revealed that therewere more intermolecular interactions in the SB3 than in theSB4 tetramer. The substituted charged residues on the surfaceare involved in interactions with adjacent molecules in a differentway, forming a new crystal packing pattern. Received June 9, 2003; revised September 11, 2003; accepted September 16, 2003.     

Chemical classes of atmospheric aerosol particles at a rural site in Central Europe during winter

Ambient atmospheric particles were studied at an ecosystem research site in the Fichtelgebirge mountains in Central Europe by single-particle analysis and bulk impactor measurements. Fuzzy clustering analysis of mass spectra of individual aerosol particles allowed chemical classification of the atmospheric aerosol. During the campaign, inorganic salts, mineral particles, and carbonaceous particles, with varying degrees of secondary components, were identified. These chemical classes exhibited preferential size ranges leading to a characteristic pattern of relative abundances with respect to particle size. A more detailed analysis revealed that 65–80% of all particles were assigned almost exclusively to one chemical class. These particle populations are assumed to be externally mixed with respect to the identified chemical classes. The temporal variations of the ratio of nitrate to ammonium (ranging between 0.37 and 0.81) determined by both impactor measurements and single-particle analyses were in good agreement. Through Monte-Carlo-type simulations, confidence intervals of the mean NO₃⁻/NH₄⁺ ratio were calculated for sub-samples of the total particle population.     

The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases

What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pK_a of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site—the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.     

Abundance and size distribution of HULIS in ambient aerosols at a rural site in South China

HUmic-LIke Substances (HULIS) comprise a significant fraction of the water-soluble organic aerosol mass and influence the water uptake properties of aerosols in the atmosphere. In this work, the abundance and size distributions of HULIS in ambient aerosols were measured in a rural location in South China at a time with a visible presence of crop residue burning. PM_2.5 samples of fresh smoke from burning rice straw and sugar cane leaves were also collected and analyzed for HULIS and major aerosol constituents. HULIS were abundant in both ambient samples and in fresh biomass burning emissions, accounting for ～60% of the water-soluble organic carbon in the ambient aerosols and ～30% in the fresh biomass burning aerosols. In the particles in the range of 0.32–1.8 μm, the abundance of HULIS was 40–90% of the combined abundance of sulfate and ammonium, suggesting that HULIS should be considered when quantifying the role of sulfate aerosols serving as cloud condensation nuclei. The size distribution of HULIS was characterized by a dominant droplet mode with a mass median aerodynamic diameter (MMAD) in the range of 0.63–0.87 μm, accounting for 81% of the total HULIS mass, a minor condensation mode (12%, MMAD: 0.23–0.28 μm) and a coarse mode (7%, MMAD: 4.0–5.7 μm). The small amount of HULIS in the coarse mode indicated that soil-derived HULIS was a very minor source. On the basis of the size distribution characteristics, HULIS were postulated to have multiple sources, including secondary formation in cloud droplets, secondary formation through heterogeneous reactions or aerosol-phase reactions, and primary emissions from biomass burning.     

Heterogeneous catalysis rate parameters in a gradientless reactor by the method of moments

The transient behaviour of an internally-recycled catalytic reactor has been studied accounting for the contributions from intraparticle diffusion, adsorption and surface reaction. Equations for the zeroth, first and second absolute moments of the break-through curves are given in terms of the rate constants for the three elementary steps (first order processes are assumed throughout). The moments can be obtained from experimental break-through curves and the rate parameters for adsorption and surface reaction then evaluated from such data. Simplified equations also are derived for the case of insignificant chemical reaction and this case is used to test the method experimentally. Values of the hydrogen adsorption equilibrium constant for γ-alumina supported Co Mo-Ni catalyst are reported.     

醛与胺均相催化合成三级酰胺研究进展

酰胺类化合物是材料科学、化学生物学及药物化学中重要的有机化合物。醛容易得到且毒性较小,出于原子经济性考虑是合成酰胺的合适原料。近年来,随着绿色化学的兴起,利用醛作为酰化试剂,通过C—H键活化方式合成酰胺键的方法得到广泛关注。研究方法中,有机小分子、过渡金属、稀土盐或配合物的催化起到重要作用,纳米催化剂的催化在酰胺键形成方法的研究也有突破。纳米粒子应用于醛与胺合成三级酰胺的反应有金属胶体纳米催化剂(准均相型)和磁性非均相型。非均相催化剂的优势在于其较高的回收使用率,易与产物分离,便于回收再利用。磁性纳米材料与以往的均相催化剂相比,分离和回收更加简单方便,重复利用率更高,但催化剂表征受到限制。探索制备方法简单、操作步骤方便和催化效果显著的普通多相纳米催化剂是未来应用于催化醛合成酰胺的新领域。综述近年来利用醛作为酰化试剂,通过C—H键活化的方式,与铵盐、二级胺、三级胺和酰胺反应合成三级酰胺化合物的方法,并总结相关反应机理。     

Applications of heterogeneous catalysis in the direct oxidation of hydrocarbons in a solid-oxide fuel cell

This review article provides an overview of our recent studies of the direct oxidation of hydrocarbons in solid-oxide fuel cells. The use of a thermally stable, highly porous, yttria-stabilized zirconia (YSZ) matrix, which allows for the optimization of the anode composition and catalytic properties, is described. Studies of the direct oxidation of hydrocarbons using anodes composed of mixtures of YSZ, copper, ceria and samaria-doped ceria are also presented. The results of these studies demonstrate that copper-ceria based anodes are active for the direct electrocatalytic oxidation of a wide range of hydrocarbons including alkanes, alkenes, and aromatics.