title: Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding.
abstract: The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) [Matthews et al. (1989) J. Mol. Biol. 205, 449-454] has implicated the glutamic acid residue at position 58 in a mechanistic role which could involve the interaction of its gamma-carboxyl side chain with the nucleotide substrate and/or the folate cofactor. The site-specific mutagenesis of Glu-58 to Gln-58 in E. coli TS provided the opportunity to explore its functional role in activity and binding. When profiled by the spectrophotometric and tritium release assays, the 370- and 760-fold decreases, respectively, in kcat and the elevated Km values for the Gln-58 mutant enzyme indicated a significant involvement of Glu-58 in substrate binding and turnover. The apparent dissociation constant for the covalent FdUMP-enzyme binary complex was 30 microM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation constants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 60-fold higher, respectively, than those for the wild-type enzyme under saturating conditions. The extent of covalent FdUMP binding to the Gln-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory ternary complex but only from 0.7 to 0.5 per dimer in the binary complex of the Gln-58 enzyme. The usual 2.1-fold enhancement of FdUMP binding to wild-type TS in the presence of CH2H4folate was not observed for the Gln-58 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208.
authors.forename: J W
authors.initials: JW
authors.lastname: Zapf
authors.forename: M S
authors.initials: MS
authors.lastname: Weir
authors.forename: V
authors.initials: V
authors.lastname: Emerick
authors.forename: J E
authors.initials: JE
authors.lastname: Villafranca
authors.forename: R B
authors.initials: RB
authors.lastname: Dunlap
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D013940:Thymidylate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005971:Glutamates; D008024:Ligands; D005973:Glutamine; D018698:Glutamic Acid; D013940:Thymidylate Synthase