Thymidylate synthase (TS) is an essential enzyme for DNA synthesis and is a target for anticancer and antimicrobial drugs. The major goal of this proposal is to understand the structural correlates of TS function, which will ultimately improve efforts to design more potent and specific inhibitors of the enzyme or use as drugs. The roles proposed for certain amino acid residues in TS, based upon its known three- dimensional structure, will be tested by making site-directed mutant variants of the enzyme and quantitating their kinetic and structural differences with the wild type enzyme. Random mutagenesis will be used to identify additional residues involved in catalysis and in nucleotide and folate binding, and residues that are essential for the structural integrity TS. This information will be essential to understanding the extensive conformational changes which TS undergoes during catalysis. X-ray diffraction studies of certain variants of TS will be performed in collaboration with colleagues at the University of California, San Francisco. Parallel mutagenesis studies will be conducted on another enzyme, dCMP hydroxymethylase (CH), which catalyzes a reaction which is similar but distinct from that catalyzed by TS. The strategy is to compare the structural correlates of the functions of these two enzymes, in order to understand the stereochemical factors that determine the catalytic specificity of each. The amino sequence of CH, an essential enzyme for DNA synthesis by bacteriophage T4, indicates that CH and TS are structural homologs. The initial site-directed mutagenesis experiments on CH, the three-dimensional structure of which is unsolved, will be based on the presumed structural homology between CH and TS. Attempts will be made to crystallize CH, for x-ray structural study in collaboration with colleagues at the University of Oregon, Eugene. Kinetic approaches previously used to unravel the TS mechanism will be employed with CH to test the hypothesis that the two enzymes share certain mechanistic features, and to reveal aspects of their mechanisms that differ. The kinetic studies will in addition provide quantitative monitors, which already exist for TS, of the changes which are effected by mutagenesis of CH. The comparison of TS and CH will reveal basic principles of enzyme design.
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