Complete Restoration of Activity to Inactive Mutants of Escherichia coli Thymidylate Synthase: Evidence that E. coli Thymidylate Synthase is a Half-the-Sites Activity Enzyme

Abstract

Escherichia coli thymidylate synthase (TS) is a dimeric protein containing identical subunits. When R126E, an inactive mutant of this enzyme, was incubated at room temperature with other inactive mutants of E. coli, TS enzyme activity gradually reappeared. The rate of activity restoration was dependent on the mutant employed. In the case of C146W, an active site mutant, the half-time required for maximal activity restoration was about one hour, which was about 500-fold faster than that obtained with C146S. The final specific activity of the mutant mixtures, based on the concentration of R126E, was equivalent to that of the wild-type TS (WT-TS). However, when the activities of E. coli WT-TS and mutant TS mixtures were compared for their extents of renaturation following denaturation as described for Lactobacillus casei TS [Pookanjanatavip, M., Yuthavong, Y., Greene, P. J., & Santi, D. V. (1992) Biochemistry 31, 10303-10309], only about one-half of the activity of WT-TS was restored, implying that the denaturation-renaturation procedure was less efficient than allowing the native TS mutant dimers to exchange subunits. If, as proposed, subunit exchange is responsible for the observed restoration of activity to the E. coli mutant TS mixtures, it would suggest that only one active site cysteine, that provided by R126E in the dimer (R126E)-(C146W), is sufficient to yield the same kcat as WT-TS, which contains one active site cysteine in each subunit. Other mutant dimers that contain both active site cysteines such as (R126E)-(Y94A) and (R126E)-(I264Am) are also fully active, even though one of the subunits is functionally inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

“…These results imply that the K282E/R283E TS-dead mutant is unable to form the covalent dUMP⅐CH 2 H 4 -folate⅐enzyme complex requisite properly for chemistry to take place. Restoration of Activity to Assess Global Stability-From studies in E. coli, it is known that TS, which exists as a dimer in most species, exhibits half-the-sites activity, meaning that at any given time, only one half of the TS dimer is kinetically competent (31,32). In E. coli TS it was shown that the amino acid Arg-126 participates in the catalytic site of the opposite half of the dimer and that a TS dimer in which both Arg-126 residues have been mutated to glutamic acid is TS-dead.…”

“…In E. coli, one TS-dead mutant whose activity can be restored by heterodimerization is K48E, homologous to K282E in L. major (31). We hypothesized that if Lys-282 and Arg-283 were crucial to global stability of the protein, then dissociating the TS dimer and reassociating the K282E/R283E mutant with an active site mutant may not lead to restoration of TS activity.…”

“…In E. coli, mutation of the residue homologous to K282E alone resulted in a TS-dead enzyme, but no mechanism of inactivation was reported (31). It was predicted that the K282E/R283E mutation would prohibit folate binding at the TS site based on the crystal structure of L. major TS-DHFR (Fig.…”

Role of Y94 in Proton and Hydride Transfers Catalyzed by Thymidylate Synthase

“…The forward commitment C f is described by equation 7: [7] where [B] is the concentration of the second substrate (CH 2 H 4 folate in this case). As apparent from Eq 6 and Eq 7, the observed T (V/K) is dependent on the concentration of B and its observed value can change between two finite values (C f changes from k 9 /k 5 to k 9 /(k 5 + k 4 ) as [B] changes from infinity to zero).…”

Escherichia coli thymidylate synthase expression protects human cells from the cytotoxic effects of 5-fluorodeoxyuridine more effectively than human thymidylate synthase overexpression

“…Kevin McDonagh and Philip Stetson (University of Michigan). For pLNCX.eTS, the eTS gene was amplified by polymerase chain reaction using pBTAH 18 as a template as well as primers that added EcoRI and XhoI sites to the 5Ј end of the gene and EcoRI and ClaI sites to the 3Ј end of the gene. The modified eTS fragment was then ligated into pUC18 (Life Technologies) at the EcoRI cloning site.…”