Structural aspects of the inhibition and catalytic mechanism of thymidylate synthase.

Abstract

Thymidylate synthase (TS) is a target for anticancer drugs, due to its unique role in the biosynthesis of an essential DNA precursor. The X-ray structures available for several bacterial enzymes have been used to design novel inhibitors of TS, to structurally analyze the binding mode of existing inhibitors, and to propose catalytic roles for amino-acid residues on the protein. The first part of this paper describes some aspects of structure-based drug design, including a recent result from the groups of Montfort and Maley emphasizing the importance of conformational changes in inhibitor binding. The second part of the paper describes the work of the author on the TS mechanism, especially the catalytic roles of active site amino acids Asn177 and Glu58 in TS from Escherichia coli. An important function for Glu58 is proposed to be preventing the excessive stabilization of a covalent intermediate. The use of isotope effects to probe the mechanistic basis for stimulation of E. coli TS by magnesium ions, and to identify differences between the E. coli and human enzymes, is described. The hypothesis that N5 of tetrahydrofolate provides the basicity for deprotonation of the nucleotide is also discussed.