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J Biol Chem, Vol. 273, Issue 40, 25809-25817, October 2, 1998
From the Departments of Pathology and Biochemistry, The Joseph
Gottstein Memorial Cancer Research Laboratory, University of Washington
School of Medicine, Seattle, Washington 98195-7705
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.
Random Sequence Mutagenesis and Resistance to 5-Fluorouridine in
Human Thymidylate Synthases
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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