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.
Mittelstaedt
D M
DM
Schimerlik
M I
MI
D001665:Binding Sites; D003854:Deoxyribonucleotides / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008433:Mathematics; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism*
D003854:Deoxyribonucleotides; D003856:Deoxyuracil Nucleotides; D005468:Fluorodeoxyuridylate; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase