DE NOVO PURINE NUCLEOTIDE BIOSYNTHESIS

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The long term goal of this research is to define the mechanisms for metabolic regulation of de novo purine nucleotide synthesis in higher eukaryotes. This will require the isolation and characterization of enzymes which catalyze key steps in de novo purine nucleotide biosynthesis. Isolation of the corresponding cDNAs and genes will also be necessary. This is now possible because of recently developed strategies to clone cDNAs which encode the purine biosynthetic enzymes from higher eukaryotes. This proposal focuses on the primary regulatory enzyme in the de novo pathway, glutamine PRPP amidotransferase. Experiments are described to clone the human glutamine PRPP amidotransferase cD4A. This cDNA will be used to investigate feedback regulation and for comparison with the enzyme in cells from hyperuricemic patients. Two key structural elements, an 11 amino acid propeptide and an uncharacterized Fe component will be investigated in the recently cloned avian amidotransferase. The working hypothesis is that these components which are conserved in bacteria and vertebrates may have important roles in catalysis and/or regulation. The gene for the avian amidotransferase will be cloned in order to complete the structure of the mRNA 5' end and to evaluate whether the GC-rich 5' untranslated mRNA functions to modulate enzyme synthesis.