Mol Pharmacol 2000 Feb;57(2):359-66
Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.
Fantz C, Shaw D, Jennings W, Forsthoefel A, Kitchens M, Phan J, Minor W, Lebioda L, Berger FG, Spencer HT.
Department of Chemistry, Columbia, South Carolina, USA.
Drug-resistant variants of thymidylate synthase (TS) can potentially
be used in gene therapy applications to decrease the
myelosuppressive side effects of TS-directed anticancer agents or to
select genetically modified cells in vivo. Mutations of proline 303 of
human TS confer resistance to TS-directed fluoropyrimidines and
antifolates
(). We generated the corresponding variants in Escherichia coli TS
(ecTS),
position 254, to better understand the mechanism by which mutations at
this residue confer resistance. In addition, because ecTS is
intrinsically
resistant to several antifolates when compared with human TS, we
suspected
that greater resistance could be achieved with the bacterial enzyme.
The
P254L enzyme conferred >100-fold resistance to both raltitrexed and
5-fluoro-2'-deoxyuridine
(FdUrd) compared with wild-type ecTS. Four additional mutants (P254F,
P254S,
P254G, and P254D), each of which complemented growth of a TS-deficient
cell line, were generated, isolated, and characterized. Steady-state
values
of K(m) for dUMP and k(cat) were not substantially different among the
variants and were comparable with the wild-type values, but K(m) for
methylenetetrahydrofolate
(CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and
k(off)
for nucleotide binding, which were obtained by stopped-flow
spectroscopy,
were virtually unchanged among the mutants. Drastic differences were
observed
for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than
observed
with the wild-type enzyme; surprisingly, the proposed isomerization
reaction
that is very evident for the wild-type enzyme is not observed with
P254S.
The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i)
values obtained for three TS-directed inhibitors. These results show
that
mutations at Pro-254 specifically affect the initial binding
interactions
between enzyme and cofactor and also alter the ability of the mutant
enzymes
to undergo conformational changes that occur on ternary complex
formation.
The crystal structure of P254S was determined at 1.5 A resolution and
is
the most precise structure of TS available. When compared with
wild-type
TS, the structure shows local conformational changes affecting mostly
Asp-253;
its carbonyl is rotated approximately 40 degrees, and the side chain
forms
an ionpair with Arg-225.