SUBSTITUTION OF GLUTAMINE FOR GLUTAMIC ACID-58 IN ESCHERICHIA-COLI THYMIDYLATE SYNTHASE RESULTS IN PRONOUNCED DECREASES IN CATALYTIC ACTIVITY AND LIGAND-BINDING

The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) [Matthews et al. (1989) J. Mol. Biol. 205, 4 49-454] has implicated the glutamic acid residue at position 58 in a m echanistic role which could involve the interaction of its gamma-carbo xyl side chain with the nucleotide substrate and/or the folate cofacto r. The site-specific mutagenesis of Glu-58 to Gln-58 in E. coli TS pro vided the opportunity to explore its functional role in activity and b inding. When profiled by the spectrophotometric and tritium release as says, the 370- and 760-fold decreases, respectively, in k(cat) and the elevated K(m) values for the Gln-58 mutant enzyme indicated a signifi cant involvement of Glu-58 in substrate binding and turnover. The appa rent dissociation constant for the covalent FdUMP-enzyme binary comple x was 30 muM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation co nstants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 6 0-fold higher, respectively, than those for the wild-type enzyme under saturating conditions. The extent of covalent FdUMP binding to the Gl n-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory te rnary 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 t he Gln-58 enzyme. The F-19 NMR spectrum of the enzyme-FdUMP binary com plex formed with the Gln-58 enzyme was similar in chemical shifts but less intense than the spectrum of the same complex formed with the wil d-type enzyme. The fluorine resonance ascribed to the enzyme:FdUMP:H4f olate complex was not only less intense but also more deshielded with the mutant enzyme than with the wild-type enzyme. Finally, a character istic resonance at -12.5 ppm attributed to FdUMP that is covalently at tached to enzyme and CH2H4folate in the inhibitory ternary complex was not observed with the mutant enzyme. These results indicated that the mutation greatly impaired folate binding but that nucleotide binding was less perturbed.