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Stabilization of the Inactive Conformation of Human Thymidylate Synthase.

Thymidylate synthase (TS) is a homodimer of approximately 72 kDa which catalyzes the reductive methylation of 2-deoxyuridine monophosphate (dUMP) using 5,10-methylenetetrahydrofolate ([CH.sub.2][H.sub.4]folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate ([H.sub.2]folate). Because this enzymatic reaction provides the sole de novo intracellular source of dTTP for DNA biosynthesis, it has been an attractive target for the development of antineoplastic and antiparasitic drugs. Primary sequence analysis has revealed that TS is one of the most highly conserved proteins known. Recent crystallographic studies in this laboratory have indicated that human TS (hTS) exists in two states, active and inactive, in solution. Based on crystallographic data, it is predicted that the inactive conformer of hTS is stabilized by phosphate anions; this prediction is supported by studies of phosphate-dependent effects on enzyme fluorescence. Phosphate binding to arginine at position 163 (R163) is predicted to be an essential interaction that stabilizes the inactive conformer. Using site-directed mutagenesis techniques, R163 was mutated to a lysine (R163K), which is the corresponding residue in rat and mouse TSs, respectively, and a threonine (R163T), which is the corresponding residue in E. coli TS. The mutant proteins have been expressed as recombinant proteins in a TS-deficient mutant strain of E. coli. We are examining the effect of substitution at position 163 on catalytic activity, on phosphate-dependent intrinsic fluorescence, and on the crystal structure of hTS. These studies may lead to the design of novel approaches for targeting TS in human tumors.
Saphronia R. Johnson, Sondra H. Berger
Department of Basic Pharmaceutical Sciences
College of Pharmacy
University of South Carolina, Columbia

Jason Phan, Lukasz Lebioda
Department of Chemistry and Biochemistry
University of South Carolina, Columbia
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Author:Johnson, Saphronia R.; Berger, Sondra H.; Phan, Jason; Lebioda, Lukasz
Publication:Bulletin of the South Carolina Academy of Science
Article Type:Brief Article
Geographic Code:1USA
Date:Jan 1, 2001
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