Effects of mutation at arginine-218 residue on the reaction of synthetic thymidylate synthase and 5-fluorouracil

Abstract

The importance of Arg-218 residue for the catalytic activity of thymidylate synthase chemically synthesized based on the amino acid sequence of thymidylate synthase from Lactobacillus casei has been studied by cassette mutagenesis and chemical modification with the arginine-specific dicarbonyl reagent phenylglyoxal. A series of mutations were constructed and functionally acceptable substitutions were confirmed by genetic complementation of thymidylate synthase deficient cells. The mutants were further characterized by determination of kinetic parameters using 5-fluoro-2'-deoxyuridylate as an active site titrant. Analysis of the mutants by genetic complementation and kinetic studies showed that Arg-218 could be essential for enzyme activity. The kcat/Km values of the inactive mutants were undetectable or much lower than those of wild type. SDS-PAGE analysis and activity measurements of crude extracts prepared from each of the mutants showed that the absence of catalytic activity is not due to a lack of production or stability of the different mutants. The results indicate that Arg-218 is critical for substrate binding or catalytic activity of thymidylate synthase.