Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases.
Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.
Landis
D M
DM
Departments of Pathology and Biochemistry, The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington School of Medicine, Seattle, Washington 98195-7705, USA.
Loeb
L A
LA
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017422:Sequence Analysis, DNA; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics*; D014529:Uridine / Q000031:analogs & derivatives* / Q000494:pharmacology
D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C001943:5-fluorouridine; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D014529:Uridine