Deoxythymidine monophosphate (dTMP) synthase (TS) is an essential enzyme for thymine and, hence, DNA synthesis. It catalyzes the formation of dTMP from deoxyuridine monophosphate (dUMP), and requires as a cofactor 5,10-methylenetetrahydrofolate (CH2THF). The reaction is thought to proceed via Michael addition of an enzymic thiol to the pyrimidine of the substrate, to form a transient covalent intermediate. The ability of TS to form stable covalent and non-covalent complexes with various inhibitors has been exploited in the design of anticancer chemotherapeutic schemes. Studies using several of these complexes have also provided valuable information about the enzyme mechanism. Most important for the understanding of the mechanism has been the ternary complex of TS with the substrate analog 5-fluoro-dUMP (FdUMP) and CH2THF. This complex is considered to be a facsimile of a proposed catalytic intermediate in which enzyme, substrate, and cofactor are linked by covalent bonds. The structures of TS complexes have previously been studied primarily by various spectroscopic methods. Now crystals exist of native TS, the binary complex of TS with the substrate dUMP, and a ternary complex of TS with substrate and a cofactor analog. The crystals of native TS and of the binary TS.dUMP complex diffract to high resolution (greater than 2.4A). Crystals of the ternary complex are currently too small for routine single crystal X-ray diffraction analysis. We propose to determine the crystal structures of native TS and binary complexes of TS with dUMP and dUMP analogs. We also propose to optimize conditions for crystallizing ternary complexes of TS with the ultimate aim of determining the crystal structure of the TS.dUMP.CH2THF complex. The results of these analyses will illuminate stereochemical details of the enzymatic reaction and provide a more rational basis for the future design of inhibitors to be used in anticancer chemotherapy.
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