A HYDROXYL GROUP AT RESIDUE-216 IS ESSENTIAL FOR CATALYSIS BY HUMAN THYMIDYLATE SYNTHASE

Structural analyses of bacterial thymidylate synthases (TSs) implicate a serine residue corresponding to Ser216 in human TS in hydrogen bond networks that are involved in binding of the nucleotide substrate, 2' -deoxyuridylate (dUMP), and that stabilize a beta-bulge in the protein . Utilizing site-directed mutagenesis, 12 mutant proteins were created with substitutions at residue 216. DNA complementation studies utiliz ing a TS-negative bacterial strain revealed that only one mutant, Thr2 16 TS, supports the growth of the bacteria in the absence of thymidine . Kinetic characterization of the mutant proteins revealed that all TS s except Thr216 TS exhibited k(cat)/K(m)s for dUMP that are 10(3)-10(4 ) times lower, relative to that of wild-type TS. In addition, Thr216 T S was the only mutant to bind the mechanism-based inhibitor, 5-fluoro- 2'-deoxyuridylate (FdUMP), into a ternary complex. Ligand binding stud ies revealed that K(d)s for dUMP binding to two defective mutants, Ala 216 and Leu216 TSs, are 12-16-fold higher than that of wild-type TS. T he data are consistent with the hypothesis that serine at this relativ e position is involved in dUMP binding; however, the data indicate tha t Ser216 has effects on catalysis, in addition to effects on dUMP bind ing. Catalysis is initiated by nucleophilic attack of the active site cysteine of TS on dUMP. The reaction rates of cysteine residues with t he sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) were slower for Ala216 TS than for wild-type TS.