Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-hydroxymethyl-dCMP (Hm5CMP) from dCMP and methylene tetrahydrofolate (CH2THF), analogous to the reaction between dUMP and CH2THF catalyzed by thymidylate synthase (TS), an enzyme of known structure. The amino acid sequence identity between invariant TS residues and CH is at least 50%. Most of the residues which contact the dUMP and CH2THF in TS are conserved in CH. It is hypothesized that CH is homologous to TS in both structure and mechanism. The project described in this thesis tests this hypothesis. In-vitro studies on catalysis by CH variants. The roles of three residues in catalysis by CH have been tested using site-directed mutagenesis. Conversion of Cys148 to Asp, Gly or Ser decreases CH activity at least 105 fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of bound dUMP to the side chain of an Asn; the corresponding CH residue is Asp179. Conversion of Asp179 in CH to Asn reduces kcat/KM for dCMP by 104 fold and increases kcat/KM for dUMP 60 fold, changing the nucleotide specificity of the enzyme. Other studies have shown that the specificity of TS was changed from dUMP to dCMP by conversion of the appropriate Asn to Asp. Based on the crystal structure of TS, a Glu residue (also conserved in CH) is proposed to catalyze formation of the N5 iminium ion methylene donor by protonation of N10 of CH2THF. In CH and TS, overall turnover and tritium exchange are tightly coupled. Replacement of Glu60 in CH or Glu58 in TS uncouples these catalytic steps. Conversion the Glu60/58 to Gln or Asp results in a 5-50 fold decrease in the ability to catalyze tritium exchange, consistent with an inability to catalyze formation of the N5 iminium ion, but also results in a 104-105 decrease in product formation. This suggests that Glu60/58is also involved in a step in catalysis after nucleotide and folate binding and proton removal from carbon 5 of the nucleotide. Isotope effect studies. The observed value of the α-secondary tritium inverse equilibrium isotope effect (EIE = 0.8) on formation of the complex between FdUMP, CH2THF and both wild-type CH and CH(D179N) indicates that carbon 6 of FdUMP is sp3 hybridized (tetrahedral) in the ternary complex. This is consistent with the hypothesis that that carbon 6 is bonded to Cys148 in the complex. Removal of Cys148in CH prevents complex formation with FdUMP. Lack of an observed α-secondary tritium kinetic isotope effect (KIE) for position 6 of dCMP for both enzymes suggests that the intrinsic KIE is masked by other rate-limiting steps or that rehybridization follows the first irreversible step. An observed KIE on carbon 6 of dUMP by CH(D179N) suggests the rate-limiting steps for the two nucleotide substrates is different. In-vivo studies catalysis by CH variants. In order to prevent recombination between CH deficient T4 phage and plasmid borne copies of CH variants, the gene coding for CH, gene 42, was deleted from the T4 chromosome. The T4Δ42 phage requires wild-type CH expressed from a plasmid to kill their host cell. CH variants C148G, D179N, E60Q, and E60D, all which exhibit at least 2000 fold lower activity in vitro, do not complement the T4Δ42 phage in vivo. Interchanging the functional domains of CH and TS. It is proposed that shortening the C-terminal loop seen in the structure of TS changes the solvent structure of the CH active-site such that it becomes more hydrated. Differences in the solvent structure of the active-site may account for differences in the catalytic specificity between CH and TS, respectively, hydration versus reduction. In order to test the hypothesis that these catalytic differences between TS and CH lie within the C-terminal portion of the enzyme, the N-terminus of the CH(D179N) variant was fused to the C-terminus of the wild-type TS to create a chimeric CH/TS enzyme. The chimeric enzyme was predicted to have specificity for dUMP and a active-site solvent structure similar to that for wild-type TS. However, the resulting protein cannot be overproduced to significant levels and does not have any detectable TS activity in vivo.