The explanation of high specificity of discriminating optical isomers in enzymatic reactions by the molecular anvil model of enzyme

The molecular anvil model of enzyme is proposed and applied to explain high specificity of discriminating optical isomers in enzymatic reactions. The molecular anvil is a mechanism which can accumulate energy from two interacting molecules and produce locally a high energy spot called anvil site. Two conditions neccessary for formation of the molecular anvil are described. For a pair of enzyme and substrate molecules these two conditions are considered to be satisfied. Assuming proper shapes and sizes for molecules of optical isomers and a hole on the surface of the enzyme molecule into which the optical isomers can fit and also assuming Lenard-Jones 12-6 type potential for each pair of interacting molecular sites. The amount of energy accumulated at the anvil site is calculated. Following the assumption that the total reactivity is determined by binding process and chemical process in which the accumulated energy at the anvil site is utilized to enhance the reaction, total reactivities for L- and D-isomers are calculated and the values of specificity of discriminating L-isomer from D-isomer are derived for various values of interaction energy. It is shown that the molecular anvil plays an important role in elevating specificity as well as producing high catalytic power of enzyme.     

Systemic approach in the physical and structural chemistry of enzyme catalysis: From primary structure to elementary event

The state of the art in enzyme catalysis is considered in terms of physical and structural chemistry. The main chemical kinetic and structural approaches are presented that can provide detailed information concerning the elementary processes making up the multistep catalytic cycle of molecular conversion at the active site of an enzyme. It is demonstrated that knowledge of the sequence of amino acids in a protein is sufficient to reconstruct the tertiary structure of this protein, to identify the catalytic groups, and to elucidate the molecular mechanism of catalysis. This approach is based on highly efficient information and computational technologies. The architecture of the active sites of enzymes is analyzed, including geometric invariants and the characteristic bond distances and angles of catalytic groups. The template method for identifying catalytic sites in the protein 4D structure is considered. The potential of molecular mechanics in the study of active sites is illustrated by the example of computer-simulated mutagenesis. Quantum chemical calculations applied to elementary events of the catalytic cycle are considered as a physical basis for understanding the catalytic mechanism and the origin of the efficiency and specificity of enzymes.     

纳米花型酶-无机杂化固定化酶研究进展

酶是一种绿色高效的生物催化剂,被广泛地应用于工业生产中,为了更好的提升游离酶的性能,酶固定化技术应运而生。然而,与游离酶相比,固定化酶活性下降以及传质受限一直是酶固定化技术亟待解决的关键问题。作为一种新型酶固定化技术,纳米花型酶-无机杂化固定化酶因具有高比表面积、高酶活性和高催化效率,且制备简单,绿色无污染受到广泛关注。本文综述了近年来纳米花型酶-无机杂化固定化酶的研究进展,根据纳米花型酶-无机杂化固定化酶的形成特点,将其分为单酶纳米花、双酶纳米花和负载型纳米花。阐述了纳米花型酶-无机杂化固定化酶的制备过程和形成机理并对纳米花型酶-无机杂化固定化酶在食品工业和检测领域的应用进展做出总结。最后,对纳米花型酶-无机杂化固定化酶的发展前景做出展望。     

环番作为人工受体和人工酶的研究进展

本文综述了水溶液和有机溶液中环番作为人工受体包结有机分子的行为,重点综述了功能化环番作为人工酶的研究进展。     

Ribonucleotide activation by enzyme ribonucleotide reductase: understanding the role of the enzyme

This article focuses on the first step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This corresponds to the activation of the substrate. In this work a large model of the active site region involving 130 atoms was used instead of the minimal gas phase models used in previous works. The ONIOM method was employed to deal with such a large system. The results gave additional information, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme in this step. Enzyme-substrate interaction energies, specific transition state stabilization, and substrate steric strain energies were obtained. It was concluded that the transition state is stabilized in 4.0 kcal/mol by specific enzyme-substrate interactions. However, this stabilization is cancelled by the cost in conformational energy for the enzyme to adopt the transition state geometry; the overall result is that the enzyme machinery does not lead to a rate enhancement in this step. It was also found that the substrate binds to the active site with almost no steric strain, emphasizing the complementarity and specificity of the RNR active site for nucleotide binding. The main role of the enzyme at the very beginning of the catalytic cycle was concluded to be to impose stereospecifity upon substrate activation and to protect the enzyme radical from the solvent, rather than to be an reaction rate enhancement.     

Catalytic,theoretical, and biological investigation of an enzyme mimic model

Artificial catalyst studies were always stayed at the kinetics investigation level, in this work bioactivity of designed catalyst were shown by the induction of biomineralization of the cells, indicating the possible use of enzyme mimics for biological applications. The development of artificial enzymes is a continuous quest for the development of tailored catalysts with improved activity and stability. Understanding the catalytic mechanism is a replaceable step for catalytic studies and artificial enzyme mimics provide an alternative way for catalysis and a better understanding of catalytic pathways at the same time. Here we designed an artificial catalyst model by decorating peptide nanofibers with a covalently conjugated catalytic triad sequence. Owing to the self-assembling nature of the peptide amphiphiles, multiple action units can be presented on the surface for enhanced catalytic performance. The designed catalyst has shown an enzyme-like kinetics profile with a significant substrate affinity. The cooperative action in between catalytic triad amino acids has shown improved catalytic activity in comparison to only the histidine-containing control group. Histidine is an irreplaceable contributor to catalytic action and this is an additional reason for control group selection. This new method based on the self-assembly of covalently conjugated action units offers a new platform for enzyme investigations and their further applications. Artificial catalyst studies always stayed at the kinetics investigation level, in this work bioactivity of the designed catalyst was shown by the induction of biomineralization of the cells, indicating the possible use of enzyme mimics for biological applications.     

RNA cleavage by a DNA enzyme with extended chemical functionality.

In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.     

An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling

DNA enzymes are single-stranded DNA molecules with catalytic capabilities that are isolated from random-sequence DNA libraries by "in vitro selection". This new class of catalytic biomolecules has the potential of being used as unique molecular tools in a variety of innovative applications. Here we describe the creation and characterization of an RNA-cleaving autocatalytic DNA, DEC22-18, that uniquely links chemical catalysis with real-time fluorescence signaling capability in the same molecule. A trans-acting DNA molecule, DET22-18, was also developed from DEC22-18 that behaves as a true enzyme with a k(cat) of approximately 7 min(-1)-a rate constant that is the second largest ever reported for a DNA enzyme. It cleaves a chimeric RNA/DNA substrate at the lone RNA linkage surrounded by a closely spaced fluorophore-quencher pair-a unique structure that permits the synchronization of the chemical cleavage with fluorescence signaling. DET22-18 has a stem-loop structure and can be conjugated with DNA aptamers to form allosteric deoxyribozyme biosensors.     

Current State of [Fe]-Hydrogenase and Its Biomimetic Models

[Fe]-hydrogenase, the third type of natural hydrogenase, is capable to heterolytically activate hydrogen molecule and transfer the resulting hydride to an unsaturated substrate, making it a promising hydrogenation catalyst. Over the last three decades, fruitful results on this enzyme have been achieved. In this review, we have summarized the major progresses about this enzyme including its structural characterisation, catalytic mechanism, cofactor biosynthesis, mimetic model development as well as artificial enzymes construction. In the meanwhile, challenges and opportunities of this enzyme and its mimetic systems in the application of synthetic chemistry and others are discussed.     

Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N¹‐methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate‐determining hydrogen‐atom abstraction on competitive σ‐ and π‐pathways on a quintet spin‐state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen‐bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.     

A new enzyme model for enantioselective esterases based on molecularly imprinted polymers

An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.     

计算机辅助糖苷酶分子设计与改造研究进展

糖苷酶作为一种重要的生物催化剂,在工业生物催化领域有着广阔的应用前景。但天然糖苷酶存在催化活性低、热稳定性和底物选择性差等缺点,严重限制了它在规模化生产中的推广应用。近年,有关糖苷酶催化机制与结构功能关系的研究备受关注,特别是计算机辅助酶设计在相关研究领域发挥着越来越重要的作用。本文综述了糖苷酶分子设计改造过程中应用的计算机辅助方法：包括同源比对、分子对接以及动力学模拟;系统阐述了这些计算方法在糖苷酶的结构与功能关系解析、酶催化分子机制、酶催化性能改造方面的应用现状。通过对上述方法的深入分析可以预见,计算机辅助方法将成为糖苷酶分子设计改造的重要手段,并且开发智能精准的计算分析方法将成为加快酶分子定向改造的新发展趋势。     

Release of enzyme strain during catalysis reduces the activation energy barrier

Several mechanisms have been considered as principal factors in enhancing the catalytic reaction velocity of enzymes: approximation, covalent catalysis, general acid-based catalysis, and strain. Among them, the strain on the substrate and/or the enzyme is often found to be brought about on association of the substrate and the enzyme. If this strain is released in the transition state, it contributes to enhancing the k(cat) value, although it does not change the k(cat)/K(m) value. In aspartate aminotransferase, however, we found by analysis of the Schiff base pK(a) values that the unliganded enzyme carries a strain in the protonated Schiff base formed between the coenzyme pyridoxal phosphate and a lysine residue. This bond is cleaved in most of the reaction intermediates, including the transition state. As a result, the activation energy between the free enzyme plus substrate and the transition state is decreased by 16 kJ/mol, equal to the value of the strain energy. The net effect of this strain is enhancement (10(3)-fold) of the catalytic efficiency in terms of k(cat)/K(m), the more important indicator of the catalytic efficiency at low concentration of the substrate.     

分子印迹模拟酶的制备与应用研究

分子印迹模拟酶是应用分子印迹技术合成的对目标分子具有特异性催化活性的聚合物,具有良好的化学和物理稳定性、结构预定性以及实用性。本文主要介绍了分子印迹模拟酶的构建策略,包括印迹过渡态类似物、印迹底物或底物类似物和其他构建途径;探讨了分子印迹模拟酶的制备方法,总结了分子印迹模拟酶在催化反应方面的应用,涉及有机合成催化、食品安全危害物分解、环境污染物降解和临床医学检验等。     

Dihydrofolate reductase: structural aspects of mechanisms of enzyme catalysis and inhibition

The mechanism of catalytic reduction of folic and dihydrofolic acids to tetrahydrofolate, which proceeds under the action of dihydrofolate reductase and the coenzyme NADPH, is considered. The roles of the enzyme active site, the coenzyme, individual amino acid residues of the enzyme, and water molecules in the catalytic reaction are discussed. Interactions of the enzyme with competitive inhibitors many of which are widely used in medicine as antitumor and antibacterial drugs are examined. The factors controlling the selectivity of inhibitor binding to bacterial forms of the enzyme are analyzed. The results of X-ray diffraction and NMR spectroscopic studies of the structures of the enzyme and its complexes with the substrate and inhibitors are surveyed. The role of specific interactions and molecular motions of the protein and ligands in the mechanism of catalysis and in the binding of the ligands to the enzyme is discussed.     

Catalytic activity of glucose oxidase after dielectrophoretic immobilization on nanoelectrodes

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, for example, antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability of the immobilization method for sensing or research in general, we want to test it for other enzymes, too. In this study, glucose oxidase (GOX) from Aspergillus niger was immobilized on TiN nanoelectrode arrays by DEP. Fluorescence microscopy showed the intrinsic fluorescence of the immobilized enzymes flavin cofactor on the electrodes. The catalytic activity of immobilized GOX was detectable, but a fraction of less than 1.3% of the maximum activity that was expected for a full monolayer of immobilized enzymes on all electrodes was stable for multiple measurement cycles. Therefore, the effect of DEP immobilization on the catalytic activity strongly depends on the used enzyme.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Catalytic hyperbranched polymers as enzyme mimics; exploiting the principles of encapsulation and supramolecular chemistry

Nature uses the principles of encapsulation and supramolecular chemistry to bind and orientate substrates within active catalytic sites. Over the years, synthetic chemistry has generated a number of small molecule active site mimics capable of catalysing reactions involving bound substrates. Another approach uses larger molecules that better represent an enzymes globular structure. These molecules mimic an enzymes structure by incorporating binding/catalytic sites within the globular structure of the polymer. As such, the electronic and steric properties around the binding/catalytic site(s) can be controlled and fine-tuned. One class of polymer that is particularly adept at mimicking the globular structure of enzymes are dendritic polymers. This review will concentrate on the use of hyperbranched polymers as synthetic enzyme mimics.     

Wild-type and molten globular chorismate mutase achieve comparable catalytic rates using very different enthalpy/entropy compensations

The origin of the catalytic power of enzymes with a meta-stable native state,e.g.molten globular state,is an unsolved challenging issue in biochemistry.To help understand the possible differences between this special class of enzymes and the typical ones,we report here computer simulations of the catalysis of both the well-folded wild-type and the molten globular mutant of chorismate mutase.Using the ab initio quantum mechanical/molecular mechanical minimum free-energy path method,we determined the height of reaction barriers that are in good agreement with experimental measurements.Enzyme-substrate interactions were analyzed in detail to identify factors contributing to catalysis.Computed angular order parameters of backbone N–H bonds and side-chain methyl groups suggested site-specific,non-uniform rigidity changes of the enzymes during catalysis.The change of conformational entropy from the ground state to the transition state revealed distinctly contrasting entropy/enthalpy compensations in the dimeric wild-type enzyme and its molten globular monomeric variant.A unique catalytic strategy was suggested for enzymes that are natively molten globules:some may possess large conformational flexibility to provide strong electrostatic interactions to stabilize the transition state of the substrate and compensate for the entropy loss in the transition state.The equilibrium conformational dynamics in the reactant state were analyzed to quantify their contributions to the structural transitions enzymes needed to reach the transition states.The results suggest that large-scale conformational dynamics make important catalytic contributions to sampling conformational regions in favor of binding the transition state of substrate.