Abstract
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.
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