| Abstract
| - Structural analyses of bacterial thymidylate synthases (TSs)implicate a serine residuecorresponding to Ser216 in human TS in hydrogen bond networks that areinvolved in binding of thenucleotide substrate, 2‘-deoxyuridylate (dUMP), and that stabilize aβ-bulge in the protein. Utilizingsite-directed mutagenesis, 12 mutant proteins were created withsubstitutions at residue 216. DNAcomplementation studies utilizing a TS-negative bacterial strainrevealed that only one mutant, Thr216TS, supports the growth of the bacteria in the absence of thymidine.Kinetic characterization of themutant proteins revealed that all TSs except Thr216 TS exhibitedkcat/Kms for dUMP thatare 103−104times lower, relative to that of wild-type TS. In addition, Thr216TS was the only mutant to bind themechanism-based inhibitor, 5-fluoro-2‘-deoxyuridylate (FdUMP), into aternary complex. Ligand bindingstudies revealed that Kds for dUMP binding totwo defective mutants, Ala216 and Leu216 TSs, are 12−16-fold higher than that of wild-type TS. The data are consistentwith the hypothesis that serine at thisrelative position is involved in dUMP binding; however, the dataindicate that Ser216 has effects oncatalysis, in addition to effects on dUMP binding. Catalysis isinitiated by nucleophilic attack of theactive site cysteine of TS on dUMP. The reaction rates of cysteineresidues with the sulfhydryl reagent5,5‘-dithiobis(2-nitrobenzoic acid) were slower for Ala216 TS thanfor wild-type TS.
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