Abstract:
: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 utilizing a TS-negative bacterial strain revealed that only one mutant, Thr216 TS, supports the growth of the bacteria in the absence of thymidine. Kinetic characterization of the mutant proteins revealed that all TSs except Thr216 TS exhibited kcat/Kms for dUMP that are 10(3)-10(4) times lower, relative to that of wild-type TS. In addition, Thr216 TS was the only mutant to bind the mechanism-based inhibitor, 5-fluoro-2'-deoxyuridylate (FdUMP), into a ternary complex. Ligand binding studies revealed that Kds for dUMP binding to two defective mutants, Ala216 and Leu216 TSs, are 12-16-fold higher than that of wild-type TS. The data are consistent with the hypothesis that serine at this relative position is involved in dUMP binding; however, the data indicate that Ser216 has effects on catalysis, in addition to effects on dUMP binding. Catalysis is initiated by nucleophilic attack of the active site cysteine of TS on dUMP. The reaction rates of cysteine residues with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) were slower for Ala216 TS than for wild-type TS.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Williams AW,Dunlap RB,Berger SHdoi
10.1021/bi972562+subject
Has Abstractpub_date
1998-05-19 00:00:00pages
7096-102issue
20eissn
0006-2960issn
1520-4995pii
bi972562+journal_volume
37pub_type
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