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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。有机溶剂中的酶催化在将手性引入关键的生物学相关化合物中的有用性已得到广泛认可。然而，此类应用中仍然存在重大缺陷，阻碍了这些生物催化剂充分发挥其潜力。特别的缺点是在非水条件下观察到的酶活性较低（与其天然水介质相比），并且缺乏酶选择性和对映选择性的可预测性。因此，试错法仍然是实现所需产品结果的最有效方法。非水介质中酶活性降低与多种因素（底物的去溶剂化、酶的灵活性和 pH 依赖性）以及结构扰动、催化三联体残基的电离状态以及有机溶剂中酶可能的聚集有关。所有这些参数取决于用作介质的有机溶剂，并在较小程度上取决于酶制备的模式。类似地，酶的选择性和对映选择性也依赖于溶剂，并且主要归因于其灵活性和结构完整性（有机溶剂塑造酶的灵活性及其结构）。过去 4 年我们对该领域的贡献包括 (a) 一种激活酶的新方法； (b) 枯草杆菌蛋白酶的结构完整性和对映选择性之间关系的证据； (c) 确定了对酶结构有害的溶剂；(d) 我们展示了灵活性和活性之间的关系，(e) 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蛋白酶嘉士伯在有机溶剂中不稳定，正如我们最初认为的那样。该提案的目标是确定、分析和理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，目前的知识水平无法轻易回答这个简单的问题，即为什么嘉士伯枯草杆菌蛋白酶在四氢呋喃中比在乙腈中更具活性和对映选择性。这一知识空白将由所提出的研究来填补，特别是由于其范围和多学科特征，结合了实验和理论方法。将在实验和理论水平上详细研究以下领域：（a）有机溶剂中酶的结构完整性，（b）悬浮酶粉末形态的变化，因为它可能与其在非水介质中的活性和稳定性有关，以及（c）质子交换机制和纯酶催化反应的活性位点咪唑（丝氨酸蛋白酶）的作用 有机溶剂。以下具体目标的实现将满足本研究的主要目标。 - 研究影响酶对映选择性的不同因素并确定每个因素的相对贡献。为了实现这一目标，将对跨越因子-对映选择性性质空间的酶-底物系统进行一系列理论计算和实验。 - 研究酶粉末的形态如何受到其悬浮的有机溶剂的影响，以及这与酶在该介质中的活性和稳定性有何关系。将使用分形分析和扫描电子显微镜 (SEM) 来表征悬浮酶的形态。 - 确定不同有机溶剂中酶的低活性是否与活性位点组氨酸的酸度/碱度有关。这将涉及：（a）使用核磁共振波谱，（b）对活性位点中的质子改组进行建模，以获得势能曲线并将其与不同溶剂中可能发生的 pKa 变化联系起来，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。 - 通过动力学和质谱、荧光、圆二色性和漫反射红外研究有机溶剂中酶失活的机制。 - 研究在有机溶剂中激活和稳定酶的新方法。