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 their 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 regulation of the very important glycolytic rate-limiting enzyme, phosphofructokinase (PFK) as well as identification and characterization of a phosphofructo-2-kinase (PFK-2). The study will also pursue the chemical mechanism of an important mitochondrial enzyme, NAD-malice enzyme. The studies on the ascarid PFK will the development of a physiological assay for the enzyme. This assay will be based on the levels of known effectors of the PFK and will be involved with correlating the activity of the enzyme in vitro that which can calculated to occur in vivo. The primary effectors studied will be AMP, fructose-2,6- bisphosphate and covalent phosphorylation of the PFK. The next set of studies will be on the mechanism and structure of the ascarid PFK. Studies on the mechanism of the enzyme will be conducted on the PFK that has been desensitized by derivatization by reaction of several histidine residues with diethylpyrocarbonate (d-PFK). They will involve studies on the kinetic mechanism of the enzyme using the techniques of isotope partitioning and positional isotope exchange. Regulatory mechanism studies will use the same effectors as those specified above. This study will measure the precise influence of the effectors on various rate processs of the enzyme. The chemical mechanism of the d-PFK will be probed with the use of pH and residue studies in order to identify groups involved in binding and catalysis. Structural studies will be conducted using amino acid sequencing of peptides form the residue studies above in order to identify areas of the PFK involved in the active and regulatory sites. Spectral studies will be conducted with UV- visible spectrophotometry, native tryptophan fluorescence and circular dichroism studies in order to probe the conformational changes taking place during binding of substrates and effectors. Tryptic studies will also be conducted to probe the overall structure of the enzyme. PFK-2 will be isolated and purified from the muscle of the ascarid. Physico-chemical studies will be carried out on the enzyme as well as studies of its ability to be stimulated by phosphorylation. PFK-2 will also be tested for fructose-2.6-bisphosphatase activity. Finally, kinetic studies on the NAd-malic enzyme will be carried out on the reverse reaction, the reductive carboxylation of pyruvate by CO2 and NADH.
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