Stopped-flow studies of 2'-deoxynucleotide binding to thymidylate synthase

The mechanism of 2'-deoxynucleotide binding to Lactobacillus casei thymidylate synthase was studied using stopped-flow kinetic techniques to monitor the decrease in intrinsic protein fluorescence upon complex formation. The data were consistent with a two-step mechanism involving a rapid preequilibrium step to form the enzyme-2'-deoxynucleotide complex followed by a slow isomerization step. Rate and equilibrium constants were determined for the three 2'-deoxynucleotides (2'-deoxyuridylate, 2'-deoxythymidylate, and 5-fluoro-2'-deoxyuridylate) as a function of temperature. Similar free energy changes were found for all 2'-deoxynucleotides; however, the enthalpy and entropy changes for each step of the reaction differed for each 2'-deoxynucleotide. The thermodynamic profiles indicated that the isomerization step stabilized the enzyme-2'-deoxynucleotide complex by an additional 1500 cal/mol.