水生动物谷胱甘肽硫转移酶研究进展

谷胱甘肽硫转移酶(gluthione S-transferase,GST)是一种广泛存在于各种生物组织中的小分子水溶性蛋白.GST属于Ⅱ相代谢酶,主要功能是催化某些内源性或外来有害物质的亲电子基团与还原型谷胱甘肽的巯基结合,增加其疏水性使其易于排出体外,同时某些GST还具有谷胱甘肽过氧化酶活性.本文着重介绍了近年来国内外对水生动物GST的基础研究包括组织分布、同工酶类型和结构基因研究,以及各种环境污染物对水生动物GST的影响,探讨其作为生物标志物的可行性.     

水生动物谷胱甘肽硫转移酶研究进展

谷胱甘肽硫转移酶（gluthione S-transferase，GST）是一种广泛存在于各种生物组织中的小分子水溶性蛋白．GST属于Ⅱ相代谢酶．主要功能是催化某些内源性或外来有害物质的亲电子基团与还原型谷胱甘肽的巯基结合，增加其疏水性使其易于排出体外，同时某些GST还具有谷胱甘肽过氧化酶活性．本文着重介绍了近年来国内外对水生动物GST的基础研究包括组织分布、同工酶类型和结构基因研究．以及各种环境污染物对水生动物GST的影响，探讨其作为生物标志物的可行性．     

基于生物新陈代谢中酶的高效催化性研究

酶是生物新陈代谢的保障条件之一,在生物的新陈代谢过程中,主要起到催化生物化学反应的作用,从而保证新陈代谢顺利进行.酶的催化作用具有高效性的特点,而这一特性又取决于酶的高效催化机理.本文从酶改变化学反应历程,与底物的结合理论及结合途径方面阐述了生物新陈代谢中酶的高效催化性.     

铽-钛铁试剂络合物荧光猝灭法测定辅酶Ⅱ

结合蛋白酶是由酶蛋白和非蛋白质部分(包括辅酶及金属离子)组成的全酶,单纯的酶蛋白没有催化功能,只有全酶才具有催化性能.     

生物大分子开放研究实验室

一、实验室概况生物大分子开放研究实验室设于中国科学院生物物理研究所,是由国家计划委员会和中国科学院共同投资建立的国家重点实验室。它主要从事生物大分子结构与功能的基础研究和应用基础研究,近期主要研究方向包括酶的催化和调控原理,酶的动力学与不可逆抑制动力学,生物大分子(包括酶、功能蛋白、多肽激素和核酸等)的空间结构、构象运动及其与功能的关系和以膜脂—膜蛋白的相互作用为中心的生物膜的结构与功能的研究。该实验室注重联系医学、农业等实际,开展应用基础研究,不断开拓研究成果的应用前景。     

蛋白质结构与动力学的计算机模拟：从方法到应用

计算机模拟已发展为根据生物分子结构和动力学阐释生物功能的重要工具.同现有实验比较,计算机模拟不仅能提供结构的时空平均,而且可获得任意微观量的时空分布和演化轨迹.除结构之外,生物功能往往依赖于对动力学的控制.计算机模拟可用于重构构象跃迁路径,发现中间体和过渡态等.本文总结了作者实验室在这一领域的近期工作,特别是关于能量模型、酶催化模拟、构象空间采样等.     

石斛碱生物合成途径及萜类化合物CYP450酶的研究进展

为了对石斛碱生物合成途径进一步解析,对石斛碱生物合成途径研究进展、5 种药用萜类生物 合成的 CYP450 酶功能、CYP450 酶研究手段三个方面进行文献综述。 通过综述石斛碱生物合成研究进展, 提出 CYP450 酶在石斛碱合成途径中羟基化中间体的作用;总结对药用萜类青蒿酸、丹参酮、鼠尾草酸、人参 皂苷、甘草甜素等合成途径中 CYP450 酶的功能研究与鉴定,发现 CYP450 酶在萜类化合物中的主要功能为 羟基化、酮基化等氧化功能;总结 CYP450 酶的研究手段:一方面基因的筛选可以通过数据库检索或植物组 织、多因子诱导表达差异筛选,另一方面基因的表达和功能鉴定通过基因工程的多种手段,转入原核或真核 宿主细胞中表达出酶蛋白,然后加入底物鉴定其催化活性。 最后,展望下一步石斛碱生物合成 CYP450 酶的 挖掘,认为可以通过已有的转录组中筛选出差异 CYP450 酶基因并进行蛋白表达,然后通过上游酶催化或从 植物中分离纯化的方式获得底物,从酶蛋白与底物的催化挖掘与石斛碱合成相关 CYP450 酶类。     

核糖核苷酸还原酶研究

核糖核苷酸还原酶广泛存在于各种生物中,是生物体内唯一的催化4种核糖核苷酸还原、生成相应的脱氧核糖核苷酸的酶。该酶是DNA合成和修复的关键酶和限速酶,对细胞的增殖和分化起着调控作用。不同生物中的RR根据其结合的金属辅助因子不同而分类。虽然不同类型RR之间的氨基酸序列相似性很低,但它们有十分相似的三级结构的活性中心和相同的催化功能。RR分子中包含2个变构位点,即酶活性中心和底物特异结合位点。活性中心通过生物有机自由基的作用催化核糖核苷酸还原;底物特异结合位点通过变构作用调控4种dNTPs在细胞内的平衡。因此,该酶不仅是研究DNA合成与修复、细胞增殖与分化及癌症的治疗与抗癌药物开发的重要靶点,同时也是研究酶的结构与功能以及酶的催化机理等的重要工具。本文总结了该酶的种类与分布、结构特征、催化机理及作为抗癌药物开发靶点等方面的研究进展。     

酶化学修饰研究的进展

前言酶是具有高效、特异催化功能的蛋白质。这种奇妙的特性既依赖于共有序的化学结构,又取决于其肽链自发折迭成的空间构象。酶的这种结构与功能关系的研究是分子生物学的中心课题,也是现代生物学研究的主要方向之一。酶的化学修饰技术是探索酶结构与功能关系的主要工具之一。所谓化学修饰就是应用化学或生物化学以及遗传学的方法使酶蛋白的化学结构共价地发生改变的技术。这种化学结构的变化不仅使酶蛋白的空间构象发生改变,而且也会影响酶的理化特性和催化功能。这样的探索在理论上有十分重要的意义,也为蛋白质工程的兴起奠定了坚实的基础。酶化学修饰的研究是酶学研究的前沿,越来越受到世界各国科学家的重视。     

人工酶与定向进化的前沿与挑战

 定向进化是在实验室环境中，在分子水平上模拟进化过程，得到具有期望特征的蛋白质的方法，目前已成为蛋白质设计改造的重要方法。定向进化不仅可以用于天然蛋白质的改造，也可以通过改造现有的酶，使其具有新的催化活性，从而构建人工酶。本文重点介绍工业生物催化、纳米酶设计和光催化3个方向的前沿成果，并讨论人工酶与定向进化领域存在的挑战和问题。     

酶改性技术研究

酶具有催化效率高、催化专一性强等显著特点,然而,酶的活力和稳定性往往不能满足应用的要求.为了改进酶的催化特性,笔者及其所在课题组20多年来长期进行酶分子修饰、酶固定化、酶非水相催化、酶定向进化等酶改性技术的研究,发现通过酶的改性,可以显著改进酶活力和稳定性.文中对笔者及其所在课题组在酶改性技术方面的研究成果和进展作了介绍.     

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.     

基因重组型枯草芽孢杆菌芽孢表面展示技术与应用

枯草芽孢杆菌是一种好氧型革兰氏阳性益生菌,在营养匮乏的环境下,可形成具有强抗逆性的芽孢。其芽孢独特的结构和生理功能使得芽孢表面展示技术相较于其他表面展示技术具有多种优势,构建的重组芽孢不但易纯化、回收率高,而且安全性好。近年来,枯草芽孢杆菌芽孢表面展示技术得到迅猛发展,已成功利用CotB、CotC、CotG、CotZ、CotA、OxdD、CotE、CotZ、CgeA等多种锚定蛋白将外源蛋白或多肽展示在芽孢表面,并应用于工业化酶、口服疫苗和药物、大分子量多聚体蛋白的生产以及环境污染的生物治理等领域。本文首先介绍了芽孢展示系统整合策略,并重点阐述了基因重组型枯草芽孢杆菌芽孢展示技术中涉及的锚定蛋白以及该技术在各领域的应用与展望。     

Enzyme catalysis: not different, just better

Where are we in our understanding of enzyme catalysis? The gloomier view is that protein structure and enzyme function are the finely balanced end-products of many weak interactions that can be summed only by massive computing power, and more precise parameterization than we enjoy at present. The cheerier position is that proteins are built on definable principles, and that enzymes use recognizable catalytic devices that will allow us to understand how existing enzymes work and to design new ones. To assess which interpretation is the more realistic, the simple reaction catalysed by triosephosphate isomerase is considered here. This examination illustrates some of the catalytic features of enzymes that are understood, and exposes a few that are not. But overall, the question turns out to have an optimistic answer.     

Designed divergent evolution of enzyme function

It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity). The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues). To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase gamma-humulene synthase. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.     

Free energy perturbation calculations on binding and catalysis after mutating Asn 155 in subtilisin

Site-directed mutagenesis is a very powerful approach to altering the biological functions of proteins, the structural stability of proteins and the interactions of proteins with other molecules. Several experimental studies in recent years have been directed at estimating the changes in catalytic properties, (rates of binding and catalysis) in site-directed mutants of enzymes compared to the native enzymes. Simulation approaches to the study of complex molecules have also become more powerful, in no small measure owing to the increase in computer power. These simulations have often allowed results of experiments to be rationalized and understood mechanistically. A new approach called the free-energy pertubation method, which uses statistical mechanics and molecular dynamics can often be used for quantitative calculation of free energy differences. We have applied such a technique to calculate the differential free energy of binding and free energy of activation for catalysis of a tripeptide substrate by native subtilisin and a subtilisin mutant (Asn 155----Ala 155). Our studies lead to a calculated difference in free energy of binding which is relatively small, but a calculated change in free energy of catalysis which is substantial. These energies are very close to those determined experimentally (J. A. Wells and D. A. Estell, personal communication), which were not known to us until the simulations were completed. This demonstrates the predictive power and utility of theoretical simulation methods in studies of the effects of site-specific mutagenesis on both enzyme binding and catalysis.     

单分子成像和追踪技术在工业微生物研究中的应用

在单分子工具和技术的迅速发展过程中,单分子荧光显微镜技术脱颖而出,可以高特异性地监测荧光标记的生物分子的时空行为,成为研究生物系统的有力工具。然而,单分子成像和追踪技术在工业微生物研究及酶工程领域中的应用仍处于起步阶段。随着生物工程技术、合成生物学和代谢工程等科学技术的迅猛发展,工业微生物的应用日趋广泛。但是,菌株往往难以在整个培养期间保持最大的生产能力,导致在经济效益上不能满足产业化的需求,成为制约微生物细胞工厂发展的一个瓶颈,对酶学研究提出新的挑战。单分子成像和追踪技术具有直接、准确、实时等监测优点,可以直接在活体生物上进行研究,成功地实现了对单分子酶催化过程的实时监控,发现了多种酶的新单分子行为及反应机制。这些技术有助于各种新型酶、新特性酶、新作用方式酶的研发。本文简述单分子成像和追踪技术的主要特点（以荧光显微镜为主）,总结近年来单分子成像和追踪技术在微生物研究、酶学研究中的应用,及其在工业微生物研究中的应用现状和面临的挑战。这些技术的应用必将促进纳米生物学的发展,推动酶工程改造,提高细胞工厂的生产能力。