STUDY OF THE UNDERLYING FACTORS THAT SHAPE ENZYME PROPERTIES IN ORGANIC SOLVENT

有机溶剂中影响酶性质的基本因素的研究

• 批准号: 7720862
• 负责人: GABRIEL Luis BARLETTA
• 金额: $ 22.42万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720862
• 关键词: Acetonitriles; Acidity; Active Sites; Affect; Area; Catalysis; Circular Dichroism; Computer Retrieval of Information on Scientific Projects Database; Condition; Crown Ethers; Dependence; Diffuse; Enzymes; Fluorescence Spectrometry; Fractals; Funding; Goals; Grant; Histidine; Imidazole; Institution; Kinetics; Knowledge; Link; Methods; Modeling; Morphology; NMR Spectroscopy; Organic solvent product; Outcome; Pliability; Potential Energy; Powder dose form; Preparation; Property; Protons; Reaction; Relative (related person); Research; Research Personnel; Resources; Role; Scanning Electron Microscopy; Series; Serine Protease; Shapes; Solvents; Source; Structure; Subtilisin; Subtilisins; Subtilopeptidase A; System; Thinking; Triad Acrylic Resin; United States National Institutes of Health; Welts; alkalinity; aqueous; desire; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; inhibitor/antagonist; ionization; multidisciplinary; research study; tetrahydrofuran

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The usefulness of enzymatic catalysis in organic solvents in introducing chirality to key biologically relevant compounds is welt recognized. However, there are still major drawbacks in such applications which preclude the use of these biocatalysts to their full potential. Particular liabilities are the low enzyme activity observed under nonaqueous conditions (as compared to their natural aqueous medium), and the lack of predictability of the enzymes' selectivity and enantioselectivity. As a consequence, a trial and error approach remains the most effective method to achieve the desired product outcome. The reduced enzyme activity in non-aqueous media has been linked to several factors (substrate's desolvation, enzyme flexibility, and pH dependence) as well as structural perturbations, the ionization-state of the catalytic triad residues, and possible aggregation of an enzyme in organic solvents. All of these parameters depend on the organic solvent used as the medium, and to a lesser extent to the mode of enzyme preparation. Similarly, an enzyme's selectivity and enantioselectivity are also solvent dependent and have been mainly attributed to its flexibility and its structural integrity (organic solvents shape both the enzyme flexibility and its structure). Our contributions to this field during the last 4 years have included (a) a new method to activate enzymes; is (b) evidence of the relationship between the structural integrity and enantioselectivity of subtilisin; (c) identified solvents which are detrimental to an enzyme's structure;is (d) we showed a relationship between flexibility and activity, (e) showed the effect of crown ethers on structure and activity, and (f) we also demonstrated that subtilisin Carlsberg is not stable in organic solvents as first thought. The goal of this proposal is to determine, analyze, and understand the crucial parameters that decide the outcome of any reaction catalyzed by an enzyme in organic solvents. The simple question, for example, as to why subtilisin Carlsberg is more active and enantioselective in tetrahydrofuran than in acetonitrile cannot be readily answered with the current state of knowledge. This knowledge gap will be filled by the proposed research especially due to its scope and multidisciplinary character combining experimental and theoretical methods. The following areas wilt be studied in detail at the experimental and theoretical level: (a) the structural integrity of an enzyme in organic solvents, (b) changes in a suspended enzyme powder's morphology as it might relate to its activity and stability in non-aqueous media, and (c) the mechanism of proton swapping and the role of the active site imidazole (in serine proteases) of reactions catalyzed in neat organic solvents. The realization of the following specific aims will satisfy the principal goal of this research. - To study the different factors that influence enzyme enantioselectivity and to determine for each factor its relative contribution. To accomplish this, a set of theoretical calculations and experiments will be conducted on enzyme-substrate systems spanning the factor-enantioselectivity property space. - To study how the morphology of an enzyme powder is affected by the organic solvents in which it is suspended, and how this relates to the enzyme's activity and stability in this media. The morphology of the suspended enzyme will be characterized using fractal analysis and scanning electron microscopy (SEM). - To determine if the low enzyme activity in different organic solvents is related to the acidity/basicity of the active-site histidine. This will involve: (a) the use of NMR spectroscopy, (b) the modeling of the proton shuffling in the active site to obtain the potential energy curves and to relate that to the possible pKa changes that might occur in different solvents, and (c) to study the catalytic role of the active site histidine in organic solvents using a series of inhibitors. - To study the mechanism of enzyme inactivation in organic solvents by kinetic and mass spectrometry, fluorescence, circular dichroism and diffuse reflectance infra-red. - To study new methods to activate and stabilize enzymes in organic solvents.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 酶催化在有机溶剂中的有用性在将手性引入关键的生物学相关化合物中得到了认识。但是，此类应用中仍然存在主要的缺点，这些缺点排除了这些生物催化剂的全部潜力。特定的负债是在非水性条件下观察到的低酶活性（与其天然水培养基相比），并且缺乏酶选择性和对映选择性的可预测性。结果，试验和错误方法仍然是实现所需产品结果的最有效方法。非水培养基中酶活性的降低与多种因素（底物的脱溶剂，酶柔韧性和pH依赖性）以及结构扰动，催化三合会残基的电离状态以及有机溶剂中酶的酶聚集的电离状态有关。所有这些参数都取决于用作培养基的有机溶剂，并且在较小程度上取决于酶制剂的模式。同样，酶的选择性和对映选择性也是溶剂的依赖性，并且主要归因于其柔韧性及其结构完整性（有机溶剂构成了酶柔性及其结构）。在过去的四年中，我们对这一领域的贡献包括（a）一种激活酶的新方法；是（b）枯草素的结构完整性和对映选择性之间关系的证据； （c）鉴定出对酶结构有害的溶剂； IS（d）我们显示了柔韧性和活动之间的关系，（e）显示了冠状醚对结构和活性的影响，并且（f）我们还证明了枯草菌Carlsberg在有机溶液中不稳定。该提案的目的是确定，分析和理解有机溶剂中酶催化的任何反应结果的关键参数。例如，一个简单的问题是关于为什么枯草菌素卡尔斯伯格在四氢呋喃中比在乙腈中更活跃和对映选择性，因此无法以当前的知识状态轻易回答。该知识差距将由拟议的研究填补，尤其是由于其范围和多学科特征结合了实验和理论方法。 The following areas wilt be studied in detail at the experimental and theoretical level: (a) the structural integrity of an enzyme in organic solvents, (b) changes in a suspended enzyme powder's morphology as it might relate to its activity and stability in non-aqueous media, and (c) the mechanism of proton swapping and the role of the active site imidazole (in serine proteases) of reactions catalyzed in neat有机溶剂。实现以下特定目标将满足这项研究的主要目标。 - 研究影响酶对酶选择性的不同因素，并确定其相对贡献的每个因素。为此，将对跨越因子 - 隔离性特性空间的酶 - 基底系统进行一组理论计算和实验。 - 研究酶粉末的形态如何受到悬浮的有机溶剂的影响，以及这与该培养基中酶的活性和稳定性有关。悬浮酶的形态将使用分形分析和扫描电子显微镜（SEM）来表征。 - 确定不同有机溶剂中的低酶活性是否与活性位点组氨酸的酸度/碱度有关。这将涉及：（a）使用NMR 光谱，（b）活性位点中质子改组的建模，以获得势能曲线，并将其与不同溶剂中可能发生的PKA变化相关联，以及（c）使用一系列抑制剂研究活性位点组氨酸在有机溶剂中的催化作用。 - 研究通过动力学和质谱，荧光，圆形二色性和弥漫性反射率Infra-Red在有机溶剂中失活的机理。 - 研究有机溶剂中激活和稳定酶的新方法。