REGULATORY AND STRUCTURAL ANALYSIS OF UMP SYNTHASE

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UMP synthase is a multifunctional protein containing both orotate phosphoribosyltransferase (OPRTase) and orotidine-5'-monophosphate decarboxylase (ODCase). These two enzymes are the last of a sequence of six enzymes required for de novo biosynthesis of UMP, the precursor of all other pyrimidine nucleotides. UMP synthase is elevated in rapidly growing tissues and cells such as those of tumor origin and has been considered a target enzyme for chemotherapy. In contrast to tumor tissue, the genetic disease, orotic aciduria, has been described and directly results from a lack of both OPRTase and ODCase (Type I) or a lack of ODCase (Type II). Chronic uridine therapy has been shown to overcome the manifestations of the disease. Recently, a cDNA containing the coding sequence of Ehrlich ascites UMP synthase has been isolated in this laboratory. DNA sequence analysis of the cDNA will allow the amino acid sequence or the protein to be determined. By the use of the appropriate expression vectors, E. coli or yeast will be programmed to synthesize UMP synthase from the cDNA. The coding region of the cDNA will be altered by frameshift mutation or by sequential nucleolytic digestion. These DNAs will be used to program OPRTase-deficient and ODCase-deficient E. coli to produce truncated proteins which complement only one deficiency. These DNAs will be analyzed by restriction mapping and DNA sequencing to determine which amino acids of the entire amino acid sequence constitute the individual OPRTase and ODCase catalytic domains. The present purification scheme of UMP synthase yields extensively denatured enzyme due to the low intracellular source. Since expression in E. coli may lead to intracellular accumulation of greater than 15% of the expressed protein, this may enable the purification of a less-denatured protein. This protein will serve as a source for structural studies which are already planned in the laboratory. Finally, the cDNA will be used as a probe to measure the mRNA levels in cultured cells to determine (a) the cell-cycle specificity and (b) the degree of transcriptional regulation of UMP synthase by gene expression.