The overall objectives are to understand the several function of thymidylate synthase (TS), one of the most conserved of all enzymes throughout living organisms, and a paradigm for methyl transfer reactions in pyrimidine biosynthesis. TS is an important drug target since it provides the sole de novo pathway for synthesis of an essential nucleotide for DNA synthesis. The stereochemistry of the carbon-carbon bond forming methyl transfer reaction, the extensive protein structural changes which serve to sequester reactants, and the reorientation of ligands which take place during catalysis are to be defined from atomic structures of complexes of thymidylate synthase which mimic intermediates in the reaction path. Roles of individual residues and water molecules will be determined quantitatively by structure determination of variants generated by mutagenesis, in binary complex with substrate or in ternary complex with substrate and cofactor, or analogs. A highly efficient mutagenesis strategy of a gene synthesized to have optimally placed, unique restriction sites is used to generate substitutions for all other natural amino acids at a site, followed by an initial rapid screen for functional variants. The most informative variants will be purified using a high efficiency expression (-5-30% total protein in E. coli), and two step purification, and assayed quantitatively to define effects on binding and catalytic steps in the reaction. Structures will be determined by difference Fournier methods against one of eight different crystal forms solved for different structural states, highly refined, and related quantitatively to alterations in binding and kinetic rates. The role of TS in transcriptional regulation of the TS gene by its binding of mRNA will be defined at the structural level. Ligand-protein structures and binding affinities for variants will be applied to derive an empirical relationship between binding affinity and solvation, entopic, electrostatic, and van der Waals forces, with the ultimate goal of developing a scheme for accurately predicting binding affinity of modified inhibitors to this or other important drug targets. The structure of a homologous enzymes which catalyzes one-carbon transfer to pyrimidine, deoxyuridylate-hydroxymethylase will be determined. The structural basis for the difference in catalyzing transfer of hydroxide versus hydride in the reaction will be defined.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA041323-10
Application #
2090423
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1985-12-01
Project End
1998-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
10
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Newby, Zachary; Lee, Tom T; Morse, Richard J et al. (2006) The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261. Biochemistry 45:7415-28
Birdsall, David L; Finer-Moore, Janet; Stroud, Robert M (2003) The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity. Protein Eng 16:229-40
Gonzalez-Pacanowska, Dolores; Ruiz-Perez, Luis M; Carreras-Gomez, Maria Angeles et al. (2003) The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase. Protein Eng 16:607-14
Stroud, Robert M; Finer-Moore, Janet S (2003) Conformational dynamics along an enzymatic reaction pathway: thymidylate synthase, ""the movie"". Biochemistry 42:239-47
Finer-Moore, Janet S; Santi, Daniel V; Stroud, Robert M (2003) Lessons and conclusions from dissecting the mechanism of a bisubstrate enzyme: thymidylate synthase mutagenesis, function, and structure. Biochemistry 42:248-56
Fritz, Timothy A; Liu, Lu; Finer-Moore, Janet S et al. (2002) Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access. Biochemistry 41:7021-9
Kawase, S; Cho, S W; Rozelle, J et al. (2000) Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase. Protein Eng 13:557-63
Variath, P; Liu, Y; Lee, T T et al. (2000) Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants. Biochemistry 39:2429-35
Morse, R J; Kawase, S; Santi, D V et al. (2000) Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of ""effective charge balance"". Biochemistry 39:1011-20
Stout, T J; Tondi, D; Rinaldi, M et al. (1999) Structure-based design of inhibitors specific for bacterial thymidylate synthase. Biochemistry 38:1607-17

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