The overall objective of this research program is to develop a clear understanding of carbohydrate and energy metabolism in parasitic helminths. Inherent in this objective is the delineation of the regulatory steps which control the flow of carbon through these pathways. A complete description of these regulatory steps and the modulators would provide unique information on the parasite and the manner in which it relates to its environment, the host. With this information, it might be possible to design chemotherapeutic agents whose mode of action would be based on the differences between the parasite and its host. These studies will be carried out on the parasitic nematode, Ascaris suum, and will concentrate on the enzyme, phosphofructokinase (PFK). A cDNA clone has been isolated that presumably contains the full sequence of PFK. It will be sequenced and then expressed in a bacterial vector. The PFK will be used to study the residues which participate in catalysis and regulation. They will be studied by derivatization with various reagents that specifically react with that residue. Carboxyls will be derivatized with N-ethyl-5- phenylisoxazolium-3'sulfonate (Woodward Reagent K), cysteines will be modified with N-ethylmaleiimide, lysines in the ATP inhibitory site with 2',3'-dialdehyde ATP histidines with diethylpyrocarbonate, and tyrosines with tetranitromethane and N-acetylimidazole. Derivatization will be by radioactive reagents and protection from enzyme inactivation by substrates and effectors will be noted. Then the enzyme will be digested with a protease and the radioactive peptides will be isolated by HPLC and sequenced with a gas-phase sequenator. With the knowledge of the sequence obtained earlier, we will be able define those residues that participate in catalysis and regulation and thus define portions of the molecule that contain the active and regulatory sites. We will also work on the kinetic mechanism of the enzyme by studying isotope partitioning and positional isotope exchange which will give information on the rate limiting steps of mechanism. We will also work on pH studies which will define those groups that must be protonated or unprotonated for catalysis or regulation. By doing these studies combined with those chemical derivatization studies above, important residues will be distinguished. Using this information plus knowledge of other PFKs, we will mutagenize certain residues, express and isolate the mutant protein and study the effect of the mutant amino acid on catalysis or regulation. In this manner, we should be able to predict what the catalytic mechanism should be. We will also study the structure of the PFK as it relates to function. The conditions will be determined under which the tetramer dissociates into the dimers or monomers. The circular dichroic spectra of the PFK will be determined when it is in its native state versus that when it is phosphorylated. We will also note the differences in the spectra when d-PFK and pd-PFK are run. Finally, utilizing the recombinant PFK a crystallization study will be begun in which the various forms of the PFK that we have developed (n-PFK, d-PFK, pn-PFK, pd PFK, o-PFK) will be tested for their ability to crystallize in the presence substrates, products and effectors.
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