几丁质酶Hevamine的三级结构和催化机理

１．引言几丁质酶存在于各种生物中,有广泛的功能。如含有几丁质酶的细菌可以利用几丁质作为碳源和能量的来源;真菌中的几丁质酶在真菌分裂增殖过程中起着水解细胞壁的作用;在具有几丁质外骨骼的节肢动物中,几丁质酶在其蜕皮过程中发挥着重要作用;植物产生的几丁质酶...     

漆酶的性质、功能、催化机理和应用

漆酶是一种结合多个铜离子的蛋白,是铜蓝氧化酶蛋白家族的一员。本文叙述漆酶的分子结构、底物特异性及其物理化学特性,并讨论漆酶的酶促反应机理和生物学功能,包括植物漆酶参与细胞壁的形成以及漆酶与病原菌毒力的关系。本文还着重介绍了漆酶在环境生物修复方面的应用。     

漆酶的性质、功能、催化机理和应用

漆酶是一种结合多个铜离子的蛋白,是铜蓝氧化酶蛋白家族的一员。本文叙述漆酶的分子结构、底物特异性及其物理化学特性,并讨论漆酶的酶促反应机理和生物学功能,包括植物漆酶参与细胞壁的形成以及漆酶与病原菌毒力的关系。本文还着重介绍了漆酶在环境生物修复方面的应用。     

唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

测定氢酶吸氢活性的光谱分析法

固氮酶催化放氢是影响生物固氮效率的重要因素之一。经过吸氢酶吸收固氮酶释放的氢,一方面可以增加还原力来源,同时经氧化后可以消除系统内的氧,保护固氮酶免受氧伤害,从而提高固氮效率。测定氢酶吸氢的方法有多种,例如:同位素氚与水的交换法、检压法、电极法、气相层析法和光谱分析法。由于前三种方法操作较繁琐,目前国内较多的是使用气相层析法。而用光谱分析法定量地测定氢酶的吸氢活性是一种比气相法更为快速和灵敏的     

小型核酶的结构和催化机理

自然界存在的小型核酶主要有锤头型核酶、发夹型核酶、肝炎δ病毒（HDV）核酶和VS核酶.锤头型核酶由3个短螺旋和1个广义保守的连接序列组成;发夹型核酶的催化中心由两个肩并肩挨着的区域构成;HDV核酶折叠成包含五个螺旋臂（P1～P4）的双结结构;VS核酶由五个螺旋结构组成,这些螺旋结构通过两个连接域连接起来.小型核酶的催化机理与其分子结构密切相关.金属离子或特定碱基都可作为催化反应的关键成分.锤头型核酶的催化必须有金属离子（尤其是二价金属离子）参与,而发夹型核酶则完全不需要金属离子.基因组HDV核酶进行催化时要有金属离子和特定碱基互相配合.     

果胶甲酯酶的结构与功能研究进展

果胶甲酯酶(PME)是一种重要的果胶酶,其水解果胶中的甲酯基从而释放甲醇并降低果胶的甲酯化程度。目前在食品加工、茶饮料、造纸等生产工艺中有着广泛的应用前景。随着对PME的深入研究,已报道了几种不同来源的酶晶体结构,对这些已获得的晶体结构进行分析发现,PME属于右手平行β-螺旋结构,其催化残基为2个保守的天冬氨酸和1个谷氨酰胺残基,并且在催化过程中分别起到了一般酸碱、亲核试剂以及稳定中间体的作用。同时对其底物特异性进行分析,初步了解其底物与活性位点的识别机制。文中针对这几个相关方面进行了系统的综述。     

色氨酸卤化酶研究进展

能催化卤代反应的卤化酶,因其具有高效、选择性好、反应条件温和的特点受到广泛关注。其中色氨酸卤化酶是研究最多的一类酶,它的特点是可以有选择性地卤化色氨酸,主要包括氯化和溴化。而卤代化合物具有多种生物活性,在医药、化工等领域有着广泛的应用。本文主要介绍了色氨酸卤化酶的来源和种类,酶学性质及其异源表达的研究现状;重点阐述了其结构和功能之间的相互关系及催化机理;并对色氨酸卤化酶的应用以及未来发展方向进行了展望,以期为色氨酸卤化酶的开发应用提供参考。     

产甲烷古菌铁氢酶研究进展

根据活性中心金属原子的不同,氢酶主要分为镍铁、铁铁、铁氢酶三大类。铁氢酶是发现较晚、存在物种单一且结构较为特殊的一类氢酶。目前,铁氢酶仅发现于氢营养型产甲烷古菌中。该酶直接催化氢气异裂,还原产甲烷代谢途径中一碳载体四氢蝶呤的次甲基转化为亚甲基。与其他两类氢酶相比,铁氢酶不含传递电子的铁硫簇和双金属活性中心,在结构组成上有较大的差异。此外,铁氢酶活性中心的吡啶环被高度取代,活性中心铁原子直接与酰基碳成键,这些奇特的活性分子结构预示着氢酶全新的催化机制,以及古菌细胞在合成特殊结构大分子方面的特殊功能。本文总结了从1990年发现这类新型氢酶以来的相关研究,分别从氢酶的生理功能、结构特征、催化机制、成熟过程及应用研究等方面阐述铁氢酶的研究进展。     

乙酰胆碱酯酶分子研究进展

乙酰胆碱酯酶是生物体内胆碱能神经传导中的一种关键酶,它能够快速水解神经递质乙酰胆碱,保证神经信号在生物体内正常传递。乙酰胆碱酯酶结构和催化功能研究一直是生命科学的重要课题,可为研制有针对性的新药和杀虫剂提供重要理论依据。我们简要介绍了乙酰胆碱酯酶的三维晶体结构、活性位点、高效催化机制及其应用。     

Catalysing new reactions during evolution: economy of residues and mechanism

The diversity of function in some enzyme superfamilies shows that during evolution, enzymes have evolved to catalyse different reactions on the same structure scaffold. In this analysis, we examine in detail how enzymes can modify their chemistry, through a comparison of the catalytic residues and mechanisms in 27 pairs of homologous enzymes of totally different functions. We find that evolution is very economical. Enzymes retain structurally conserved residues to aid catalysis, including residues that bind catalytic metal ions and modulate cofactor chemistry. We examine the conservation of residue type and residue function in these structurally conserved residue pairs. Additionally, enzymes often retain common mechanistic steps catalyzed by structurally conserved residues. We have examined these steps in the context of their overall reactions.     

Using a library of structural templates to recognise catalytic sites and explore their evolution in homologous families

Catalytic site structure is normally highly conserved between distantly related enzymes. As a consequence, templates representing catalytic sites have the potential to succeed at function prediction in cases where methods based on sequence or overall structure fail. There are many methods for searching protein structures for matches to structural templates, but few validated template libraries to use with these methods. We present a library of structural templates representing catalytic sites, based on information from the scientific literature. Furthermore, we analyse homologous template families to discover the diversity within families and the utility of templates for active site recognition. Templates representing the catalytic sites of homologous proteins mostly differ by less than 1A root mean square deviation, even when the sequence similarity between the two proteins is low. Within these sets of homologues there is usually no discernible relationship between catalytic site structure similarity and sequence similarity. Because of this structural conservation of catalytic sites, the templates can discriminate between matches to related proteins and random matches with over 85% sensitivity and predictive accuracy. Templates based on protein backbone positions are more discriminating than those based on side-chain atoms. These analyses show encouraging prospects for prediction of functional sites in structural genomics structures of unknown function, and will be of use in analyses of convergent evolution and exploring relationships between active site geometry and chemistry. The template library can be queried via a web server at and is available for download.     

Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus lon reveals the conformational variability in the active sites of lon proteases

The atomic-resolution crystal structure of the proteolytic domain (P-domain, residues 415-621) of Archaeoglobus fulgidus B-type Lon protease (wtAfLonB) and the structures of several mutants have revealed significant differences in the conformation of the active-site residues when compared to other known Lon P-domains, despite the conservation of the overall fold. The catalytic Ser509 is facing the solvent and is distant from Lys552, the other member of the catalytic dyad. Instead, the adjacent Asp508 forms an ion pair with the catalytic lysine residue. Glu506, an analog of the putative third catalytic residue from a related Methanococcus jannaschii LonB, also faces the solvent and does not interact with the catalytic dyad. We have established that full-length wtAfLonB is proteolytically active in an ATP-dependent manner. The loss of enzymatic activity of the S509A mutant confirms the functional significance of this residue, while retention of considerable level of activity by the D508A and E506A mutants rules out their critical involvement in catalysis. In contrast to the full-length enzymes, all individually purified P-domains (wild-type and mutants) were inactive, and the mutations had no influence on the active-site structure. These findings raise the possibility that, although isolated proteolytic domains of both AfLonB and E.coli LonA are able to assemble into expected functional hexamers, the presence of the other domains, as well as substrate binding, may be needed to stabilize the productive conformation of their active sites. Thus, the observed conformational variability may reflect the differences in the stability of active-site structures for the proteolytic counterparts of single-chain Lon versus independently folded proteolytic subunits of two-chain AAA+ proteases.     

Dynamic features of cAMP-dependent protein kinase revealed by apoenzyme crystal structure

To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other protein kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.     

Functional sites in protein families uncovered via an objective and automated graph theoretic approach

We report a method for detection of recurring side-chain patterns (DRESPAT) using an unbiased and automated graph theoretic approach. We first list all structural patterns as sub-graphs where the protein is represented as a graph. The patterns from proteins are compared pair-wise to detect patterns common to a protein pair based on content and geometry criteria. The recurring pattern is then detected using an automated search algorithm from the all-against-all pair-wise comparison data of proteins. Intra-protein pattern comparison data are used to enable detection of patterns recurring within a protein. A method has been proposed for empirical calculation of statistical significance of recurring pattern. The method was tested on 17 protein sets of varying size, composed of non-redundant representatives from SCOP superfamilies. Recurring patterns in serine proteases, cysteine proteases, lipases, cupredoxin, ferredoxin, ferritin, cytochrome c, aspartoyl proteases, peroxidases, phospholipase A2, endonuclease, SH3 domain, EF-hand and lectins show additional residues conserved in the vicinity of the known functional sites. On the basis of the recurring patterns in ferritin, EF-hand and lectins, we could separate proteins or domains that are structurally similar yet different in metal ion-binding characteristics. In addition, novel recurring patterns were observed in glutathione-S-transferase, phospholipase A2 and ferredoxin with potential structural/functional roles. The results are discussed in relation to the known functional sites in each family. Between 2000 and 50,000 patterns were enumerated from each protein with between ten and 500 patterns detected as common to an evolutionarily related protein pair. Our results show that unbiased extraction of functional site pattern is not feasible from an evolutionarily related protein pair but is feasible from protein sets comprising five or more proteins. The DRESPAT method does not require a user-defined pattern, size or location of the pattern and therefore, has the potential to uncover new functional sites in protein families.