Characterization of Glucose-6-Phosphate Isomerase Activity in Organic Media

Enzyme-mediated catalysis activity has been shown to be preserved in organic solvents. The preservation of a tightly associated, often partial monolayer of water, even after lyophilization, has been suggested to be a major contributor to this activity preservation. A change in substrate specificity has also been observed in this situation. This may be due to conformational changes driven by hydrophobic/hydrophilic interactions between the enzyme, the monolayer and the bulk solvent, with subsequent surface minimization to decrease the systems energy. However, this is beyond the scope of this study. The study will examine the activity of glucose-6-phosphate isomerase in various organic solvents and with different substrates with the goal of determining optimal conditions, if they exist, to catalyze a specific organic synthesis. I. Range Finding The enzyme activity will be examined in various aqueous/solvent mixtures. Initially, these solvents will be methanol, acetone and acetonitrile. These solvents are chosen because they are associated with the targeted organic synthesis that will be studied once preliminary studies justify further examination of this system. The solvent mixtures will be 12.5%, 25%, 50% with 0% and 100% performed as controls. Activity will be assessed similarly in all solvents so that the highest level of acceptable enzyme activity can be determined. In this case, even though we would presumably see the best activity in minimal solvent mixture, we would be looking for an acceptable level of preservation of activity,e.g.10-20%, that would make the reaction useful as a catalytic tool II. Substrate Specificity Examination of the substrate specificity is important as we would required the enzyme to bind and react with compounds that are similar to its normal substrate but perhaps containing different substituents. At some higher levels of organic solvent, substrate solubility may be a problem, but this can be ameliorated by covalent modification of the molecule, e.g. fatty acid esterification or alcoholic condensation, that would increase the solubility. Given the miscibility of the chosen solvents with water, we may not expect to many problems with the substrate solubility. Examined substrates would include other hexose-6-phosphates (furanoses and pyranoses) and substituted furans and pyrans. The phosphate group of the hexose provides energy to complete the reaction so it may be necessary to supply the reaction mixture with phosphate, possibly in the form of pyrophosphate (PPi), so that the reaction is completed. The addition of a phosphate may also be mediated by some other enzyme, most likely a less specific kinase, but this has to be examined in further detail. III. Organic Catalysis Once the reaction system is identified, we can move on to the actual task of using the system to catalyze a specific organic reaction.. The biggest problem is finding a reporter system to track the reaction that correlates closely with concentration like a Beers Law plot