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.
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