The mechanism of nonstereospecific hydrogen transfer catalyzed by E. coli UDP-galactose 4-epimerase will be investigated. Association and dissociation rate constants for inhibitors and substrates will be measured. Isotope effects for reactions of deuterated substrates will be determined. One or more active site-directed alkylating agents will be synthesized and used to identify a general base at the active site. The phosphorus NMR spectra of the epimerase NAD complex and its complexes with substrates and inhibitors will be determined. The mechanism of coupling between electron and group transfer in PDH and other alpha-ketoacid dehydrogenase complexes will be investigated. Acetyl-thiamin pyrophosphate will be specifically generated at the active site of component E1 and evaluated as a prospective intermediate. The stereochemistry of decarboxylation and dehydrofluorination of 3-fluoropyruvate by PDH complex of E. coli will be determined. Possible interactions of PDH complex with the E. coli membrane will be investigated. Large, electron dense inorganic cluster complexes will be derivatized for use as specific labels for the components of alpha-ketoacid dehydrogenase complexes. The clusters may enable the visualization and specific indentification of components of multienzyme complexes by electron microscopy. These enzymes play key roles in metabolism of animal cells and bacteria, and both are related to genetic defects in metabolism.
