计算机辅助蛋白结构预测及酶的计算设计研究进展

随着不可再生资源的日益减少以及化学催化所带来的环境污染问题的日益严重,应用低污染的生物催化技术改造或取代传统化工工艺已经成为可持续发展的研究热点。酶作为一种重要的生物催化剂,具有高效无污染、专一性较强等优点,在化工、医药等领域具有巨大的应用潜力和良好的发展前景。但由于新功能酶的开发速度较慢,使其应用受到严重限制。酶的理性设计是新酶发现的一个重要来源,随着计算机技术的发展,蛋白质结构预测方法和新功能酶计算设计策略得到迅猛的发展,已成为酶功能改造的有力工具和新的研究前沿。本文就蛋白质结构预测方法、新功能酶计算设计方法和策略以及未来发展趋势进行简要介绍和讨论。     

施肥对蕹菜硝态氮累积和土壤酶活性的影响

为了研究施肥方式对蕹菜硝态氮累积和土壤酶活性的影响, 在上海郊区一蔬菜园艺场设置了6个施肥处理: 不施肥对照; 施用复合化肥量分别为900, 1 800和2 700 kg/ha; 施用菜籽饼肥量分别为2 250, 4 500 kg/ha, 并定期监测蕹菜硝态氮含量的变化和土壤酶活性的差异. 结果表明: 复合化肥的施肥量越多, 蕹菜硝态氮的累积量就越高, 且肥料施用量与蔬菜硝酸盐含量呈极显著正相关(p<0.01); 在蕹菜的整个生长周期内, 植株的硝态氮含量先上升, 至21 d左右达到最大值, 然后迅速减少, 至54 d 后趋于平稳, 且在可食用范围内; 不同施肥处理的蕹菜在生长后期, 其土壤脱氢酶、过氧化氢酶、脲酶和酸性磷酸酶的活性均显著高于施肥前的背景土壤, 说明蕹菜生长有利于增加土壤酶活性. 施菜籽饼肥的土壤酶活性显著高于施复合化肥, 说明增施有机肥可提高土壤酶活性.     

酶催化功能混杂性研究进展

酶催化生物体内化学反应,是生命代谢形成运转的动力。与传统观点认为酶具有专一性催化功能相对,近年来生物信息与实验分析都证实了酶具有多种混杂催化功能是普遍存在的现象。在过去的几十亿年里,古老酶一直不断演化以适应变化的环境,形成现代功能多样的酶蛋白家族。基于独特底物结合模式和动态蛋白结构的催化功能混杂性是酶蛋白适应性演化的基础。酶的混杂活性有望被开发应用于药物酶法合成及环境修复领域。本文就酶催化功能混杂性的普遍性、分子机理、可进化性等方面的相关研究进展进行综述。     

Intrinsic motions along an enzymatic reaction trajectory

The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.     

林地土壤酶活性季节动态

我省杉木连栽林地土壤酶活性具有显著的季节性变化,多数酶活性春季开始上升,夏、秋季达最高,冬季下降至最低;同时,土壤酶活性和土壤的营养元素含量与杉木年阶段发育有密切关系,杉木生长发育最旺盛季节,需要养分最多时期,土壤酶活性也最为活跃;土壤酶活性和土壤营养元素含量的年均动态表现为一代杉木林>二代杉木林,或三代杉木林。     

植物过氧化物酶的结构、催化反应及生理功能

过氧化物酶(EC.1.11.1.7)是一种以H_2O_2作为电子受体催化底物的氧化作用的酶,这种酶广泛存在于生物体中,这种酶已被广泛研究。本文讨论了过氧化物酶的化学结构、过氧化物酶的亚基构成、它们的催化反应及机理,同时对其的生理功能也作了较祥细的讨论。     

A conformational switch controls hepatitis delta virus ribozyme catalysis

Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.     

半干旱区土壤酶活性与其理化及微生物的关系

在半干旱典型区域选择了8个不同植物群落样地对4类土壤酶活性及与土壤理化性质、微生物量的关系进行研究.结果表明,各种酶活性受制于水分、盐碱等因素,含量均较低.大多数酶在同一样地各个剖面中变化趋势一致.相关分析表明,影响各种酶活性的土壤物理、化学、生物性状有一定的差异,其中大多与有机质含量、微生物含量有非常密切的关系.用土壤化学性质对各种酶活性进行逐步回归分析,可以得到理想的方程.主成分分析发现,徽生物量和中性磷酸酶在各主成分中均有较大的相关系数,可认为是该地区有代表性的土壤生物特性因子.另外,对土壤表层酶活性和微生物量进行聚类分析,可以将8个样地分为不同的类别.     

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.     

Intrinsic dynamics of an enzyme underlies catalysis

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.     

十字花科植物中异硫氰酸盐的性质及活性研究

对十字花科植物中的异硫氰酸盐(ITCs)的结构及性质进行分析,并对其抑制I相酶、诱导II相酶的活性及诱导肿瘤细胞凋亡使细胞周期停止等主要抗癌机制进行探讨.目前,国外已经将SFN产品上市销售,并可以治疗高血压、白内障等棘手性疾病.对异硫氰酸盐的结构性质及药理活性作以综述.     

Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation.

Enzyme-based chemical transformations typically proceed with high selectivity under mild conditions, and are becoming increasingly important in the pharmaceutical and chemical industries. Cytochrome P450 monooxygenases (P450s) constitute a large family of enzymes of particular interest in this regard. Their biological functions, such as detoxification of xenobiotics and steroidogenesis, are based on the ability to catalyse the insertion of oxygen into a wide variety of compounds. Such a catalytic transformation might find technological applications in areas ranging from gene therapy and environmental remediation to the selective synthesis of pharmaceuticals and chemicals. But relatively low turnover rates (particularly towards non-natural substrates), low stability and the need for electron-donating cofactors prohibit the practical use of P450s as isolated enzymes. Here we report the directed evolution of the P450 from Pseudomonas putida to create mutants that hydroxylate naphthalene in the absence of cofactors through the 'peroxide shunt' pathway with more than 20-fold higher activity than the native enzyme. We are able to screen efficiently for improved mutants by coexpressing them with horseradish peroxidase, which converts the products of the P450 reaction into fluorescent compounds amenable to digital imaging screening. This system should allow us to select and develop mono- and di-oxygenases into practically useful biocatalysts for the hydroxylation of a wide range of aromatic compounds.     

Industrial biocatalysis today and tomorrow

The use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Biocatalytic processes can now be carried out in organic solvents as well as aqueous environments, so that apolar organic compounds as well as water-soluble compounds can be modified selectively and efficiently with enzymes and biocatalytically active cells. As the use of biocatalysis for industrial chemical synthesis becomes easier, several chemical companies have begun to increase significantly the number and sophistication of the biocatalytic processes used in their synthesis operations.     

蛇毒的研究进展及其在医药领域的应用

就国内外主要毒蛇的蛇毒组分、对凝血系统的影响及蛇毒在医药领域的应用进行了综述．蛇毒的化学成分十分复杂,主要包括酶、神经毒性多肽、神经生长因子、膜活性多肽及其他生物活性肽,其中酶类包含多种与凝血系统、纤溶系统相关的成分．蛇毒在医药领域具有非常重要的作用,主要有促凝作用、溶栓作用、镇痛作用、降压作用和抗肿瘤作用．     

酶化学修饰的研究进展

化学修饰已成为改进酶理化性质和生物活性的有效方法 ,引起了人们高度的重视 .本文主要介绍酶化学修饰的目的、意义、方法 ,以及修饰剂的选择、种类 ,评述了酶化学修饰领域的主要进展、研究概况及其应用     

酶固定化载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一。固定化酶能在较为温和的反应条件下高选择性、高效率地催化某些化学反应,并且不会对环境造成污染。作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响所得固定化酶的催化活性及操作稳定性。综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容。综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势。     

微生物催化合成丙烯酸的研究进展

丙烯酸是一种重要的平台化合物,从它出发可以合成一系列市场广阔、附加值高的产品。传统的丙烯酸化学合成法以不可再生的化石资源为原料,并对环境造成严重污染。本文从可持续发展及环境保护的角度出发,综述丙烯酸的微生物合成途径以及合成关键酶等方面的研究进展,分析探讨不同生物合成途径的优劣之处,并展望利用微生物高产丙烯酸存在的瓶颈以及有潜力的发展方向。     

The stereochemical course of amino acid activation by methionyl- and tyrosyl-tRNA synthetases.

Stereochemical analysis has long been recognised as a powerful tool for elucidating the mechanisms of chemical and enzyme-catalysed reactions. Although much is known about the stereochemical course of reactions at saturated carbon, phosphate and thiophosphate esters whose ligands to phosphorus are also tetrahedrally disposed, are capable in principle of revealing sterochemical information about events at the active site of enzymes that transform such substrates. Nucleotidyl transferases are a group of enzymes which in general selectively use one of the diastereoisomers of a nucleoside 5'(1-thiotriphosphate), such as isomers A and B of adenosine 5'(1-thiotriphosphate), designated ATP alpha S-A and ATP alpha S-B, and allow investigation of the stereochemical course of nucleotidyl transfer. We have developed a simple method based on 31P nuclear magnetic resonance spectroscopy for determining the stereochemical course of these reactions, and using this method show here that the nucleotidyl transfer step in two aminoacyl-tRNA synthetases from Escherichia coli occurs with inversion of configuration at phosphorus. These observations greatly constrain the mechanistic possibilities for these enzymes, and are interpreted most simply as a direct 'in line' transfer from ATP to the amino acid.     

增菇激活酶对香菇生长的促进作用

在配制香菇菌袋栽培料时添加0.2%(质量分数)增菇激活酶,研究增菇激活酶对菌丝生长周期、菌丝胞外酶活力、栽培料理化指标、生物学效率及子实体营养成分的影响.实验结果表明,添加增菇激活酶组的菌丝满袋时间为70 d,比对照组提前15 d.在菌丝转色期,增菇激活酶组菌丝纤维素酶、蛋白酶和淀粉酶活力持续增加,且显著高于对照组.出菇后采收子实体,增菇激活酶组的生物学效率达96.75%,比对照组提高21.99%,子实体营养成分略高于对照组,无显著差异.     

活性氧双向生物学效应和机制

指出了活性氧（ROS）是普遍存在于有氧代谢中的产物,其化学性质极为活跃,有关ROS对人体的危害已逐渐得到公认.然而,越来越多的研究提示,一直被认为是人类疾病元凶的ROS对机体和细胞也有有益的一面.综述了ROS的研究进展,揭示了ROS通过调节离子通道、受体、酶以及转录因子的活性和改变细胞氧化还原状态,进而参与细胞的正常生理过程（细胞的增殖、分化和凋亡）,从而讨论了ROS的双向生物学效应和机制.