| The work described herein is focused on the development of amino acid analogues for applications in protein biochemistry. Particularly successful among these has been difluoromethionine, an analogue primarily intended for use in 19F NMR spectroscopy. With this compound, it was possible to address problems associated with trifluoromethionine incorporation into proteins, such as low protein yield and incomplete labelling, and it was possible to obtain quantitatively labelled protein with no apparent reduction in yield. Additionally, incorporation of this compound into bacteriophage lambda lysozyme, the E. coli leucine/isoleucine/valine binding protein, and RNase A revealed that information may be obtained about the degree of side chain packing in particular areas of the protein due to the sensitivity of the geminal fluorine atoms to rotational mobility. The ease of synthesis and very effective protein labelling will hopefully translate to the adoption of this methodology by other researchers involved in the investigation of protein structure-function relationships.; Two amino acid analogues were prepared with uniquely reactive side chains for the incorporation of novel chemical modification sites into proteins. The compounds monofluoroethionine and thioasparagine were incorporated into bacteriophage lambda lysozyme and reactions selective for each functional group were attempted on the modified proteins. It appeared that the nucleophilic labelling of monofluoroethionine-labelled LaL was successful, but the results for the thioasparagine-labelled protein were inconclusive.; In addition, it appears that in the process of incorporating monofluoroethionine into LaL, some of the amino acid appears to undergo hydrogen fluoride elimination with the end result being the incorporation of the vinyl analogue into the protein. Although it has not yet been possible to conclusively characterize the route by which this elimination occurs, structural and kinetic analyses suggest that the enzyme methionyl-tRNA synthetase may be a candidate for carrying out this reaction in vivo.; To increase the promiscuity of the biosynthetic machinery toward the selection of modified Met analogues, experiments were performed to engineer E. coli methionyl-tRNA synthetase, the enzyme thought to be the most discriminating in the incorporation pathway, to accept structurally altered Met analogues more efficiently. These efforts were unsuccessful; however, these mutagenesis experiments, along with kinetic and structural analyses of the wild-type enzyme, have dramatically increased the available knowledge concerning the key structural and dynamic features of the enzyme responsible for substrate recognition, as well as the properties of the substrate itself which are required for effective recognition and catalysis.; Finally, the knowledge gained concerning the chemistry of fluoromethyl sulfides was applied to the design of mechanism-based inhibitors for peptide methionine sulfoxide reductase and dimethyl sulfoxide reductase. Although the monofluoromethyl sulfoxides designed were unsuccessful as inhibitors of these enzymes, it may be that these compounds could display potency for other enzymes such as biotin sulfoxide reductase. In addition, tailoring the appendages of the molecules to promote greater affinity for targeted enzymes could result in the design of an effective mechanism-based inhibitor. (Abstract shortened by UMI.)... |
