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A mutant human thymidylate synthase (TS) has been created in which a glutamine residue at position 214 has been replaced by glutamate. Glutamine at position 214 is postulated to be involved in maintaining the enzyme in a conformation that facilitates the binding of the substrate dUMP. Although the kcat/Km of the mutant protein for the substrate, dUMP, is 10(3) lower than that of wild-type TS, the mutant TS confers thymidine prototrophy on a TS-deficient bacterial strain when expressed at high levels. In the present investigation, a TS-deficient Chinese hamster lung cell line was transfected with DNA encoding the defective protein. Thymidine prototrophs were isolated that expressed the defective protein at levels that were physiologically relevant. The activities of the enzymes expressed endogenously in representative prototrophs were consistent with the activities observed for the purified proteins. At similar levels of TS expression, thymidine prototrophs expressing Glu214 TS were 8-fold more resistant to 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity than are prototrophs expressing Gln214 TS. FdUrd is a prodrug of the tight-binding TS inhibitor, 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The resistance to FdUrd was associated with a significant decrease in the binding of FdUMP to the purified mutant enzyme. The data are consistent with the interpretation that TSs that are highly defective are capable of sufficient dTMP production for cell survival and optimal growth, yet may confer resistance to TS-directed inhibitors.

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)

The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

The results presented herein describe the development of a new method for the purification of thymidylate synthase from amethopterin-resistant L. casei. This method includes three chromatographic steps: batchwise CM-Sephadex, Q-Sepharose, and 10-formylfolate-Sepharose affinity steps, and the whole procedure can be performed in three days. Additionally, the procedure has consistently produced enzyme with a specific activity of 3.0 to 3.2. The first step, the batchwise purification of L. casei cell free extracts on CM-Sephadex, resulted in a 120-fold purification with 115% recovery of the activity applied to the resin. The Q-Sepharose purification step yielded a 1.5-fold purification producing enzyme with a specific activity of 2.4 units/mg. As the final step in the sequence, 10-formylfolate-Sepharose affinity chromatography yielded enzyme with a specific activity of 3.0-3.2 units/mg. The entire procedure provided a 230-fold purification with 67% recovery of the activity originally present in the cell free extract. However, the purified enzyme required extensive dialysis to achieve maximal activity. The data presented also clearly demonstrate the resolution of thymidylate synthase on the 10-formylfolate affinity column into forms which have different specific activity values and FdUMP binding ratios. Generally, enzyme with low activity leaches off this affinity matrix first, while enzyme with high activity elutes off last. These results are in accord with the sensitivity of the dimeric thymidylate synthase active site cysteines to oxidization. Consequently, when one of the two active site cysteines is oxidized, the enzyme loses the ability to bind FdUMP at this active site and its specific activity is proportionally lower. Thus, pure thymidylate synthase protein exists as a mixture of heterogeneous activity states. This condition is best examined by performing native PAGE on enzyme that has been incubated with excesses of FdUMP and CH2H4folate.