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 by all organisms that have the de novo biosynthesis of UMP, a 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 a target enzyme for chemotherapy. The genetic disease, orotic aciduria, has been described and directly results from a defective UMP synthase lacking both OPRTase and ODCase (Type I) or only ODCase (Type II). Chronic uridine therapy overcomes most of the manifestations of the disease. We have purified human UMP synthase to homogeneity by a monoclonal antibody affinity column. A human UMP synthase cDNA will be placed in an overproducing vector so that we can obtain large quantities of this protein for structure/function studies which will include X-ray crystallography, mechanism studies, and site-specific mutagenesis. We will identify amino acid residues or peptide sequences are essential for substrate/inhibitor binding, for enzyme catalysis, for the conformational states of the protein and regulation of activity. Comparative studies with the monofunctional yeast ODCase, the pure ODCase domain of mouse UMP synthase and the recombinant OPRTase and ODCase domains of human UMP synthase will allow us to assess whether there are unique characteristics in the bifunctional protein. Our overall goal is to establish whether the bifunctional protein, UMP synthase, simply represents the sum of its covalently-joined enzyme domains or whether it also represents an entity with additional kinetic and regulatory characteristics, since this protein (or therapy that can provide the normal product, UMP) is necessary for normal human growth and development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM034539-07A1
Application #
3285735
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1985-01-01
Project End
1994-11-30
Budget Start
1991-12-20
Budget End
1992-11-30
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599