Tj. Connick et al., P-31 NUCLEAR-MAGNETIC-RESONANCE STUDIES OF COMPLEXES OF THYMIDYLATE SYNTHASE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1208(1), 1994, pp. 118-126
The interactions of thymidylate synthase (TS) with deoxyuridylate (dUM
P), deoxythymidylate (dTMP) and 5-fluorodeoxyuridylate (FdUMP) were ex
amined by P-31-NMR. Single P-31 resonances appeared at 3.3 ppm, 3.2 pp
m and 3.0 ppm from the standard, 85% phosphoric acid, for unbound dUMP
, dTMP, and FdUMP, respectively. Incubation of the enzyme with either
dUMP or dTMP, alone, resulted in new resonances at 3.9 and 3.6 ppm, re
spectively, which were assigned to noncovalent complexes with the enzy
me. The same experiment employing FdUMP as the ligand gave two new res
onances appearing at 3.6 and 4.6 ppm, which were attributed to noncova
lent and covalent binary complexes, respectively. When the cofactor, C
H2H4 folate, was present in the solution with enzyme and FdUMP, a new
resonance appeared at 5.1 ppm, corresponding to the covalent inhibitor
y ternary complex. The ternary complex comprised of the enzyme, dUMP a
nd the quinazoline folate CB 3731 produced a resonance at 5.0 ppm at t
he expense of the resonance due to the enzyme-dUMP binary complex at 3
.9 ppm. Similarly, the ternary complex consisting of TS with dTMP and
CB 3731 showed a deshielding of the resonance at 3.6 ppm by 0.8 ppm. A
maximum binding of 1.5 nucleotides per enzyme dimer was found for dUM
P and dTMP in both the presence and the absence of the quinazoline fol
ate. The deshielding observed was attributed to changes in the interac
tion of the phosphate group with the nearby residues of the active sit
e of the enzyme.