Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding

• Published in: Biochemistry (Easton) 1993-09, Vol.32 (36), p.9274-9281
• Main Authors: Zapf, James W, Weir, Mary S, Emerick, Victoria, Villafranca, J. E, Dunlap, R. Bruce
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Catalysis; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Glutamates - metabolism; Glutamic Acid; Glutamine - metabolism; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Mutagenesis, Site-Directed; Thymidylate Synthase - genetics; Thymidylate Synthase - isolation & purification; Thymidylate Synthase - metabolism; Transferases
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00087a003

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.