The objective of this proposal is the use of NMR, mechanistic strategies, and immunochemical approaches to study thymidylate synthase which catalyses the 5, 10-methylenetetrahydrofolate- dependent reductive methylation of deoxyuridylate to form thymidylate. Its strategic role in thymidylate biosynthesis has make the latter enzyme an attractive target for selected pyrimidine deoxynucleotides and antifolates which serve as drugs in the treatment and management of certain cancers. In view of the expected x-ray crystallographic determination of the three- dimensional structure of Lactobacillus casei thymidylate synthase, we propose the cloning of the L. casei gene for this enzyme and the investigation of catalysis, binding, and structural aspects of the enzyme via site-directed mutagenesis. We also propose to isolate, purify, and characterize thymidylate synthase from fluorodeoxyuridine-resistant HEp-2 cells (human laryngeal carcinoma cell line) and compare its properties with those of the L. casei enzyme. Immunological studies are directed at creating enzyme-linked immunoassays of thymidylate synthase and its modified forms and at studying structure-function relationships in these proteins. Characterization of mechanistic steps in the formation of binary and ternary complexes is planned through the use of a variety of NMR and biochemical approaches.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA015645-16
Application #
3164239
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1979-04-01
Project End
1993-03-31
Budget Start
1991-04-01
Budget End
1993-03-31
Support Year
16
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of South Carolina at Columbia
Department
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Connick, T J; Reilly, R T; Dunlap, R B et al. (1994) Phosphorus-31 nuclear magnetic resonance studies of complexes of thymidylate synthase. Biochim Biophys Acta 1208:118-26
Cisneros, R J; Zapf, J W; Dunlap, R B (1993) Studies of 5-fluorodeoxyuridine 5'-monophosphate binding to carboxypeptidase A-inactivated thymidylate synthase from Lactobacillus casei. J Biol Chem 268:10102-8
Connick, T J; Reilly, R T; Dunlap, R B et al. (1993) Fluorine-19 nuclear magnetic resonance studies of binary and ternary complexes of thymidylate synthase utilizing a fluorine-labeled folate analogue. Biochemistry 32:9888-95
Bradshaw, T P; Dunlap, R B (1993) Characterization of a novel form of thymidylate synthase: a heterodimer isolated after specific chemical modification of the immobilized native enzyme. Biochemistry 32:12774-81
Bradshaw, T P; Dunlap, R B (1993) Characterization of the covalent chromatography of thymidylate synthase on thiopropyl-Sepharose 6B. Biochim Biophys Acta 1163:165-75
Zapf, J W; Weir, M S; Emerick, V et al. (1993) Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding. Biochemistry 32:9274-81
Boles, J O; Tolleson, W H; Schmidt, J C et al. (1992) Selenomethionyl dihydrofolate reductase from Escherichia coli. Comparative biochemistry and 77Se nuclear magnetic resonance spectroscopy. J Biol Chem 267:22217-23
Bradshaw, T P; Dunlap, R B (1992) Catalysis and ligand binding by thymidylate synthase immobilized on thiopropyl-sepharose 6B. Oncol Res 4:249-54
Zhang, H C; Cisneros, R J; Deng, W L et al. (1991) Purification and characterization of recombinant mouse thymidylate synthase. Biochim Biophys Acta 1077:35-46
Tolleson, W H; Alibhai, M; Cisneros, R J et al. (1991) Comparison of ELISA with activity and ligand-binding methods for the determination of thymidylate synthase concentration. Bioconjug Chem 2:327-32

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