Structure of a serpin-protease complex shows inhibition by deformation

The serpins have evolved to be the predominant family of serine-protease inhibitors in man. Their unique mechanism of inhibition involves a profound change in conformation, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical serpin-protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the serpin. This cleaves the reactive centre, which then moves 71 A to the opposite pole of the serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the serpins with their selective advantage.     

Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A

The Diels-Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state. This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites. However, only four purified enzymes have thus far been implicated in biotransformations that are consistent with a Diels-Alder reaction, namely solanapyrone synthase, LovB, macrophomate synthase, and riboflavin synthase. Although the stereochemical outcomes of these reactions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate more than one catalytic activity, leaving their specific influence on the cycloaddition step uncertain. In our studies of the biosynthesis of spinosyn A, a tetracyclic polyketide-derived insecticide from Saccharopolyspora spinosa, we identified a cyclase, SpnF, that catalyses a transannular [4+2] cycloaddition to form the cyclohexene ring in spinosyn A. Kinetic analysis demonstrates that SpnF specifically accelerates the ring formation reaction with an estimated 500-fold rate enhancement. A second enzyme, SpnL, was also identified as responsible for the final cross-bridging step that completes the tetracyclic core of spinosyn A in a manner consistent with a Rauhut-Currier reaction. This work is significant because SpnF represents the first example characterized in vitro of a stand-alone enzyme solely committed to the catalysis of a [4+2] cycloaddition reaction. In addition, the mode of formation of the complex perhydro-as-indacene moiety in spinosyn A is now fully established.     

Diels-Alder反应在斑蝥素衍生物合成中的应用

斑蝥素作为传统中药斑蝥的主要有效活性成分,在治疗肿瘤方面发挥重要作用.在对斑蝥素进行合成和改造过程中,Diels-Alder反应被广泛应用.以呋喃及其衍生物为双烯体,炔烃、马来酸酐为亲双烯体,两组分在回流或室温下就能发生成环反应获得斑蝥素基本骨架.以几类不同底物发生Diels-Alder反应制备斑蝥素衍生物并对实验条件进行了优化.     

Structural insights into the stereochemistry of the cyclooxygenase reaction

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.     

前线轨道对环加成反应的择向效应──1,4;2,1规则的作用

Diels-Alder反应通常是以一些反应熵减少,通过协同机理进行的_π4_s+_π2_s的环加成反应。然而,这种环加成反应的择向效应却很少报道.本文根据前线轨道能量及其原子轨道系数的变化,首次提出了称之谓“1-4;2-1”的规则,使环加成反应的择向问题规律化,在实际应用中简便易用,一目了然。     

铬离子催化铈离子氧化甲酸的双核配合物反应机理

在酸性介质中用氧化还原滴定法研究了铈(Ⅳ)离子在铬(Ⅲ)离子催化作用下,于30～45 ℃区间氧化甲酸的反应动力学.结果表明反应对铈(Ⅳ)和甲酸均为一级,准一级速率常数kobs随催化荆[Cr(Ⅲ)]增加而增大.亦随[H+]增加而增大,而随[HSO4-]增加而减小.在氮气保护下,反应能引发丙烯腈聚合,说明在反应中有自由基产生.提出了催化剂、底物和氧化荆间生成双核配合物的反应机理.通过kobs与HSO-4;的依赖关系.并结合Ce(Ⅳ)在溶液中的平衡,认为本反应的动力学活性物种是Ce(SO4)2,还计算出一些速率常数及相应的活化参数.     

改性磷矿渣稳定铅污染土的赋存形态及浸出特性试验研究

采用改性磷矿渣(MPS)对铅(Pb)污染土进行稳定化处理.基于浸出试验、形态提取试验和矿物分析试验,研究了改性磷矿渣(MPS)添加量对稳定土中Pb的浸出特性、赋存形态和矿物成分的影响规律,并与相同添加量的普通硅酸盐水泥(OPC)作对比.结果表明：MPS和OPC均可降低污染土中Pb的浸出特性,但MPS对Pb稳定效果明显优于OPC.与OPC稳定土相比,MPS稳定土具有更好的环境安全特性和较低的溶出风险.形态提取试验表明：OPC可促进Pb从弱酸提取态向可还原态转化,而MPS可促进Pb从弱酸提取态向可氧化态转化,污染土中Pb赋存形态的改变是MPS和OPC稳定土Pb变化的主要原因.矿物分析试验表明：OPC对Pb的稳定机制为包裹、吸附和沉淀作用,而MPS稳定对Pb的稳定机制为生成难溶的氟磷氯铅矿沉淀.铅污染土中矿物成分的变化是Pb赋存形态和浸出特性变化的根本原因.     

CaCO_3热分解的非等温动力学机制研究

本文建立了确定固态热分解非等温反应机制的动力学模型,根据该模型研究了CaCO_3分解的反应动力学。结果表明;不同升温速度条件下CaCO_3分解受不同的反应机制控制.同时;随着升温速度提高.CaCO_3分解的活化能降低。     

抗菌剂及其抗菌机理

抗菌剂包括无机、有机和天然生物抗菌剂三大类型.无机抗菌剂以新型光催化型和载银的纳米复合型抗菌材料为主要发展趋势,其中光催化型无机抗菌剂依赖光致激发的强氧化自由基而起杀菌作用;载银等金属离子型抗菌剂通过与活性基团如巯基键合或置换金属离子辅基等方式使微生物的生命活性物质失活而起抗菌作用.有机抗菌剂则以开发专效于生物分子(如微生物代谢酶、膜受体等)的抗菌剂为其拓展方向,其通过作用于细胞壁和细胞膜系统、生化反应酶、遗传物质等达到抗抑或杀菌作用.天然生物抗菌剂可来源于所有生物体,主要包括多糖、多肽及糖肽聚合物类物质,是未来抗菌材料的主要发展方向;它们作用于微生物胞外结构层或酶等生物活性物质,影响微生物的运动、跨膜物质运输或生化反应等.不同的抗菌剂对同一种病原菌有不同的抗菌作用机理和有效性,同一种抗菌剂对于不同的病原菌也有不同的抗菌作用机制和抑制范围;得到既长效又广谱、既高效又安全的抗菌剂,对其抗菌机理的研究十分重要.     

Peptide exosite inhibitors of factor VIIa as anticoagulants

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.     

塑性加工润滑添加剂协合效应的作用机制

测试了多种润滑添加剂油品的油膜承载能力,并利用Ｘ射线衍射分析了油品在金属铝表面上自然形成的吸附膜的结构,考察了油品油膜承载能力与吸附膜结构之间的关系,提出了吸附膜结构强化的概念;对复合型添加剂协会效应的作用机制进行了探讨,认为吸附膜的结构强化是引起某些复合添加剂出现协合效应的原因。     

咪唑型离子液体催化Diels-Alder反应的理论研究

采用量子化学从头计算和密度泛函理论方法,研究了咪唑类离子液体催化Diels-Alder反应(环戊二烯和丙烯酸甲酯反应)的反应机理.在HF/6-31 G*和B3LYP/6-31 G*方法水平上,考察了反应的endo/exo(内型/外型)选择性、活化能等.研究结果表明,离子液体主要借助阳离子参与来催化该反应.阳离子的参与能够大大的降低反应过渡态的能垒,增大反应的endo选择性.     

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.     

Iron metabolism in the mononuclear phagocyte system

The maintenance of body iron homeostasis requires the coordination of multiple regulatory mechanisms of iron metabolism. The mononuclear phagocyte system （MPS, composed of monocytes, macrophages, and their precursor cells） is crucial in the maintenance of iron homeostasis. Recycling of iron is carried out by specialized macrophages via engulfment of aged erythrocytes. The iron stores of macrophages depend on the levels of recovered and exported iron. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. Recent studies characterizing the function and regulation of natural resistance-associated mac- rophage protein 1 （Nrampl）, divalent metal transporter 1 （DMT1）, HLA-linked hemechromatosis gene （HFE）, ferroportin 1 （FPN1）, and hepcidin are rapidly expanding our knowledge on the molecular level of MPS iron handling. These studies are deepening our under- standing about the molecular mechanism of iron homeostasis and iron-related diseases.     

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.     

乳化法合成硫氰酸铵

对由CS_2和NH_3反应合成NH_4SCN的反应机理进行了探讨,指出该反应受扩散过程控制,并认为通过乳化方法可增加接触面积,消除扩散影响,从而使反应速率增加。为了缩短反应时间,提高生产能力,找到一种乳化剂用于合成反应过程,试验结果表明,通过引入乳化剂,可使反应时间缩短4/5,同时表明,复合乳化荆好于单一乳化剂,MOA系列和O系列乳化荆用于合成反应在各种性能方面较好。     

结构加成水解法合成二聚环戊二烯改性不饱和聚酯

制备了相关模型化合物 ,应用化学分析、NMR分析方法进行鉴定 ,提出结构加成水解法合成二聚环戊二烯改性不饱和聚酯的反应机理 .合成反应过程中 ,二聚环戊二烯降冰片环上的9、10双键与顺丁烯二酸的羧基在H+存在下发生加成反应 ,生成顺丁烯二酸—DCPD酯 .在合成反应过程中二聚环戊二烯是端基封闭剂 ,无Diels -Alder加成反应发生     

论狄尔斯-阿尔德反应

从狄尔斯-阿尔德反应的定义、机理、类型、影响因素、区域选择性及立体化学等方面进行了综合阐述.     

考虑预应力分布的立管涡激振动特性分析

针对立管结构中存在着复杂预应力分布的现象,提出了考虑复杂预应力的立管涡激振动问题.基于梁结构理论,从应力-应变方程出发,建立了含复杂预应力的立管结构运动控制微分方程.对剪切流作用下典型顶张立管的涡激振动问题进行了数值计算,对比了复杂预应力对立管结构的自然频率、振型及典型节点振动特性的影响.计算结果表明,复杂预应力的存在对立管结构的动力特性有着重要的影响,特别是结构自然频率.文中所提出的方法可为复杂载荷作用下的立管结构动力特性问题的研究提供新思路.     

MPS方法在三维溃坝问题中的应用

MPS(Moving Particle Semi-Implicit)方法是一种拉格朗日粒子法,在处理大变形流动问题中具有很大的优势.将MPS方法应用到三维溃坝流动问题中,并对粒子的移动方式进行了修改,改进后的方法(XMPS)可以使粒子移动更加有序,避免粒子间的相互穿透.数值计算结果表明:MPS方法(包括XMPS)在处理复杂的自由面流动问题中具有很好的灵活性和可靠性,数值模拟能够比较准确地预测出自由面的形状和位置,即使在水面出现翻卷、入水和水柱撞击到障碍物时,数值结果仍然能够与实验相吻合.从细节上看,XMPS可以给出更为清晰、光滑的自由面形状,并能更好地捕捉到流动的细节特征.