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)

Structure of Human Thymidylate Synthase Suggests Advantages of Chemotherapy with Noncompetitive Inhibitors

“…One unit of enzyme activity is defined as the amount of enzyme required to synthesize 1 mol of dTMP per minute. Enzyme concentration was determined by measurement of absorbance at 280 nm, as described previously (15).…”

Entropy in Bi-substrate Enzymes: Proposed Role of an Alternate Site in Chaperoning Substrate into, and Products out of, Thymidylate Synthase

“…The X-ray structure of the product complex reveals a coordination of water molecules to protein residues that is important for the catalytic conversion of dUMP to dTMP. Mutation of Glu60 greatly compromises enzymatic activity for both E. coli and Lactobacillus casei TS (Zapf et al, 1993;Huang & Santi, 1994). While distant from the nucleotide in the active site, the side-chain of Glu60 exerts its catalytic effects through the mediation of a well-ordered water molecule (labeled Wat I in Figure 4(c) located between it and the Asn229, (Finer-Moore et al, 1993) to protein of ternary complex: distances between atomic positions of equivalent atoms within each segment were added together and this sum was divided by the number of atoms within the segment.…”

Biomolecular study of human thymidylate synthase conformer-selective inhibitors: New chemotherapeutic approach

“…Proteins were visualized using 0.25% w/v Coomassie brilliant blue staining [ 17 ] ( S1 Fig ). The concentrations of purified TS proteins were determined by absorption at 280 as described previously [ 18 ]. Purified proteins were stored in 50 mM Tris-HCl, pH 7.4, containing 0.2% mercaptoethanol, 1 mM EDTA, and 15% glycerol.…”