Substitution at residue 214 of human thymidylate synthase alters nucleotide binding and isomerization of ligand-protein complexes

Based on crystal structures of bacterial thymidylate synthases (TS), a glut amine corresponding to residue 214 in human TS (hTS) is located in a region that is postulated to be critical for conformational changes that occur up on ligand binding. Previous steady-state kinetic studies indicated that rep lacement of glutamine at position 214 (Gln214) of hTS by other residues res ults in a decrease in nucleotide binding and catalysis, with only minor eff ects on folate binding (D, J, Steadman ct al, (1998) Biochemistry 37, 7089- 7095). The data suggested that Gln214 maintains the enzyme in a conformatio n that facilitates nucleotide binding, In the present study, transient-stat e kinetic analysis was utilized to determine rate constants that govern spe cific steps along the catalytic pathway of hTS, which provides the first de tailed kinetic mechanism for hTS. Analysis of the reaction mechanisms of mu tant TSs revealed that substitution at position 214 significantly affects n ucleotide binding and the rate of chemical conversion of bound substrates t o products, which is consistent with the results of steady-state kinetic an alysis. Furthermore, it is shown that substitution at position 214 affects the rate of isomerization, presumably from an open to a closed form of the enzyme-substrate complex. Although the affinity of the initial binding of C H(2)H(4)folate is not substantially affected, K-iso, the ratio of the forwa rd rate of isomerization (k(iso)) to the reverse rate of isomerization (k(r ,iso)), is 2-6-fold lower for the mutants at position 214 compared to Q214, with the greatest effects on k(iso). In addition, the binding of the folat e analogue, CB3717, to dUMP binary complexes of mutant enzymes was characte rized by a slow isomerization phase that was not detected in binding studie s utilizing wild-type hTS. The data are consistent with the hypothesis that Gln214 is located at a structurally critical region of the enzyme.