Genetic complementation and resistance to 5-fluoro-2 '-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylatesynthase
Jw. Zapf et al., Genetic complementation and resistance to 5-fluoro-2 '-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylatesynthase, BIOCH PHARM, 58(6), 1999, pp. 973-981
A mutant human thymidylate synthase (TS) has been created in which a glutam
ine residue at position 214 has been replaced by glutamate. Glutamine at po
sition 214 is postulated to be involved in maintaining the enzyme in a conf
ormation that facilitates the binding of the substrate dUMP. Although the k
(cat)/K-m of the mutant protein for the substrate, dUMP, is 10(3) lower tha
n that of wild type TS, the mutant TS confers thymidine prototrophy on a TS
deficient bacterial strain when expressed at high levels. In the present i
nvestigation, a TS-deficient Chinese hamster lung cell line was transfected
with DNA encoding the defective protein. Thymidine prototrophs were isolat
ed that expressed the defective protein at levels that were physiologically
relevant. The activities of the enzymes expressed endogenously in represen
tative prototrophs were consistent with the activities observed for the pur
ified proteins. At similar levels of TS expression, thymidine prototrophs e
xpressing Glu214 TS were 8-fold more resistant to 5-fluoro-2'-deoxyuridine
(FdUrd) cytotoxicity than are prototrophs expressing Gln214 TS. FdUrd is a
prodrug of the tight-binding TS inhibitor, 5-fluoro-2'-deoxyuridine-5'-mono
phosphate (FdUMP). The resistance to FdUrd was associated with a significan
t decrease in the binding of FdUMP to the purified mutant enzyme. The data
are consistent with the interpretation that TSs that are highly defective a
re capable of sufficient dTMP production for cell survival and optimal grow
th, yet may confer resistance to TS-directed inhibitors. BIOCHEM PHARMACOL
58;6:973-981, 1999. (C) 1999 Elsevier Science Inc.