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: 7381560
• 负责人: GABRIEL Luis BARLETTA
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381560
• 关键词: STUDY; UNDERLYING; FACTORS; SHAPE; ENZYME

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.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。有机溶剂中的酶催化在将手性引入关键的生物相关化合物方面的作用得到了广泛的认可。然而，在这类应用中仍然存在重大缺陷，这阻碍了这些生物催化剂的充分利用。特殊的缺陷是在非水条件下观察到的酶活性较低(与其自然水介质相比)，以及酶的选择性和对映体选择性缺乏可预测性。因此，试错法仍然是实现预期产品结果的最有效方法。在非水介质中酶活性的降低与几个因素(底物的脱溶、酶的灵活性和pH依赖性)以及结构扰动、催化三元残基的电离状态和酶在有机溶剂中的可能聚集有关。所有这些参数都取决于用作介质的有机溶剂，在较小程度上取决于酶的制备方式。同样，酶的选择性和对映体选择性也依赖于溶剂，主要归因于它的灵活性和结构完整性(有机溶剂塑造了酶的灵活性和结构)。我们在过去4年中对该领域的贡献包括：(A)一种激活酶的新方法；(B)枯草杆菌菌素结构完整性和对映体选择性之间关系的证据；(C)确定对酶结构有害的溶剂；(D)我们展示了柔韧性和活性之间的关系；(E)我们展示了冠醚对结构和活性的影响；以及(F)我们还证明了枯草杆菌嘉士伯酶在有机溶剂中并不像最初认为的那样稳定。这项建议的目标是确定、分析和了解决定有机溶剂中酶催化的任何反应结果的关键参数。例如，为什么枯草杆菌菌素在四氢呋喃中比在乙腈中更具活性和对映体选择性，这个简单的问题在目前的知识状况下是不容易回答的。这一知识空白将被拟议的研究填补，特别是由于其范围和多学科特征，实验方法和理论方法相结合。将在实验和理论水平上详细研究以下领域：(A)酶在有机溶剂中的结构完整性；(B)悬浮酶粉末的形态变化，因为它可能与其在非水介质中的活性和稳定性有关；(C)质子交换机制和在有机溶剂中催化反应的活性部位咪唑(在丝氨酸蛋白酶中)的作用。以下具体目标的实现将满足本研究的主要目标。-研究影响酶对映体选择性的不同因素，并确定每个因素的相对贡献。为了做到这一点，我们将对酶-底物体系进行一系列的理论计算和实验，这些体系跨越了因子-对映选择性性质空间。-研究酶粉末的形态如何受到悬浮在其中的有机溶剂的影响，以及这与酶在该介质中的活性和稳定性有何关系。用分形分析和扫描电子显微镜对悬浮酶的形态进行了表征。-确定酶在不同有机溶剂中的低活性是否与活性部位组氨酸的酸碱性有关。这将涉及：(A)使用核磁共振波谱，(B)模拟活性中心的质子洗牌，以获得势能曲线，并将其与不同溶剂中可能发生的PKA变化联系起来，以及(C)使用一系列抑制剂研究活性中心组氨酸在有机溶剂中的催化作用。-用动力学、质谱学、荧光、圆二色谱和漫反射红外光谱研究有机溶剂中酶的失活机理。-研究在有机溶剂中激活和稳定酶的新方法。