Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyses the transfer of a methyl group from methylenetrahydrofolate to dUMP to form dTMP. Based on structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop [181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197]. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor vs dUMP. In contrast, vs methylenetrahydrofolate at concentrations lower than 0.25 μM PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 μM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop [181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197] and the other in the inactive conformation.Thymidylate synthase (TS) catalyzes the reaction in which the nucleotide deoxyuridylate (dUMP) is reductively methylated by the folate co-substrate 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) to form thymidylate (TMP) and dihydrofolate (1). Substrates are bound in an ordered manner, with dUMP binding at the active site prior to CH 2 H 4 PteGlu. A cysteine residue (Cys195 in hTS) at the active site attacks the 6-position of the pyrimidine base of the nucleotide, resulting in the formation of a covalent bond between TS and the nucleotide and activating the 5-position of the nucleotide for subsequent covalent-bond formation with the C-11 substituent of CH 2 H 4 folate (reviewed in 2-4). The enzyme is the sole source of de novo synthesized thymidylate and its inhibition leads to apoptosis of rapidly dividing cells such as cancer cells, † This work was supported by NIH Grant CA 76560 and the South Carolina Cancer Center. ‡ The PDB file with atomic coordinates of hTS complex with propane-1,3-bisphosphonate have been deposited in the Protein Data Bank as entry xxxx. Data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. *To whom correspondence should be addressed at the Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter St., Columbia, South Carolina 29208. Phone (803) 777-2140; fax (803) 777-9521; e-mail lebioda@mail.chem.sc.edu. 1 Abbreviations: TS, thymidylate synthase; ...

Loop 181-197 of human thymidylate synthase (hTS) populates two conformational states. In the first state, Cys195, a residue crucial for catalytic activity, is in the active site (active conformer); in the other conformation, it is about 10 A away, outside the active site (inactive conformer). We have designed and expressed an hTS variant, R163K, in which the inactive conformation is destabilized. The activity of this mutant is 33% higher than that of wt hTS, suggesting that at least one-third of hTS populates the inactive conformer. Crystal structures of R163K in two different crystal forms, with six and two subunits per asymmetric part of the unit cells, have been determined. All subunits of this mutant are in the active conformation while wt hTS crystallizes as the inactive conformer in similar mother liquors. The structures show differences in the environment of catalytic Cys195, which correlate with Cys195 thiol reactivity, as judged by its oxidation state. Calculations show that the molecular electrostatic potential at Cys195 differs between the subunits of the dimer. One of the dimers is asymmetric with a phosphate ion bound in only one of the subunits. In the absence of the phosphate ion, that is in the inhibitor-free enzyme, the tip of loop 47-53 is about 11 A away from the active site.

Loop 181-197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k cat /K m reduced by factors in a 2-12 range. One of the mutants, M190K, is however unique in having the value of k cat /K m smaller by a factor of~7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181-197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188-194 of this loop, assumes a different conformation with the positions of C a shifted up to 7.2 Å . This affects region 116-128, which became ordered in M190K while it is disordered in wt. The conformation of 116-128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of about 27 amino acids which is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild type human TS (wt hTS) while V3T is much more stable; V3L, V3F and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with Km,app values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of loop 181–197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position which stabilizes loop 181–197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a binary complex V3F·FdUMP, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high Km,app value is caused not by stabilization of the inactive conformer but by substrate binding in a non-productive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high Km,app values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.