Structural Basis of Substrate Recognition in Thiopurine S-Methyltransferase

Abstract

Thiopurine S-methyltransferase (TPMT1) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine by methylating them in a reaction using S-adenosyl-L-methionine as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have determined two crystal structures of murine TPMT, as a binary complex with the product S-adenosyl-L-homocysteine and as a ternary complex with S-adenosyl-L-homocysteine and the substrate 6-mercaptopurine, to 1.8 Å and 2.0 Å resolution respectively. Comparison of the structures reveals that an active site loop becomes ordered upon 6-mercaptopurine binding. The positions of the two ligands are consistent with the expected S N 2 reaction mechanism. Arg147 and Arg221, the only polar amino acids near 6-mercaptopurine, are highlighted as possible participants in substrate deprotonation. To probe whether these residues are important for catalysis, point mutants were prepared in the human enzyme. Substitution of Arg152 (Arg147 in murine TPMT) with glutamic acid decreases Vmax and increases Km for 6-mercaptopurine but not Km for S-adenosyl-L-methionine. Substitution at this position with alanine or histidine, and similar substitutions of Arg226 (Arg221 in murine TPMT), result in no effect on enzyme activity. The double mutant Arg152Ala/Arg226Ala exhibits a decreased Vmax and increased Km for 6-mercaptopurine. These observations suggest that either Arg152 or Arg226 may participate in some fashion in the TPMT reaction, with one residue compensating when the other is altered, and that Arg152 may interact with substrate more directly than Arg226, consistent with observations in the murine TPMT crystal structure. with accession codes 3BGI and 3BGD, respectively. 1 Abbreviations: TPMT, thiopurine S-methyltransferase; mTPMT, murine TPMT; hTPMT, human TPMT; AdoMet, S-adenosyl-Lmethionine; AdoHcy, S-adenosyl-L-homocysteine; 6MP, 6-mercaptopurine; MAD, multiple wavelength anomalous dispersion; SAMT, salicylic acid carboxyl methyltransferase; PrmC, N 5 -glutamine methyltransferase, the product of the protein release factor methylation gene; PAPT, putrescine aminopropyltransferase; TIMP, thioinosine monophosphate; dcAdoMet, decarboxylated S-adenosyl-Lmethionine; AdoDATO, S-adenosyl-1,8-diamino-3-thiooctane. Thiopurines such as 6-mercaptopurine (6MP), 6-thioguanine, and azothioprine are cytotoxic and are immunosuppressant compounds which are used to treat childhood acute lymphoblastic leukemia, inflammatory bowel disease, and transplant rejection (1-3). They are prodrugs which are extensively metabolized to give thioguanine nucleotides that exert their cytotoxic effects by incorporation into DNA or inhibiting purine synthesis. Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme which modulates the cytotoxicity of thiopurines by taking them as substrates in an S-methylation reaction. TPMT is a classic example of pharmacogenetics, in which allelic variation in patients r...

Thiopurine S-methyltransferase - characterization of variants and ligand binding

“…Using this approach, we have characterized the kinetics of 6-MP and 6-TG methylation by TPMT. The results are in excellent agreement with the rates obtained using the radiochemical assay, and the calculated activation energy corresponds to previously reported values [28].…”

“…The reaction proceeds through an S N 2 type mechanism [9,27], where the reactive methyl group of SAM is transferred to a nucleophilic center (sulfur atom) on the 6-MP substrate, forming the products SAH and 6-Me MP. A solvent channel provides access to the active site of the enzyme, where the imidazole moiety of bound 6-MP can be oriented to either side [9,28], but formation of product should be possible regardless of the relative orientation. Studies of murine TPMT have shown that after binding of 6-MP, deprotonization of the active thiol tautomeric form is necessary to achieve an activation energy (20 kcal mol −1 ) [27] consistent with the observed reaction rate [28].…”

“…A solvent channel provides access to the active site of the enzyme, where the imidazole moiety of bound 6-MP can be oriented to either side [9,28], but formation of product should be possible regardless of the relative orientation. Studies of murine TPMT have shown that after binding of 6-MP, deprotonization of the active thiol tautomeric form is necessary to achieve an activation energy (20 kcal mol −1 ) [27] consistent with the observed reaction rate [28]. Although the precise circumstances have not been determined, deprotonization through water mediated hydrogen bonding has been proposed [9,27].…”

“…Although the precise circumstances have not been determined, deprotonization through water mediated hydrogen bonding has been proposed [9,27]. Despite the agreement between the calculated activation energy and observed reaction rates of the murine enzyme, the published kinetic parameters of human TPMT vary significantly, as does the source of protein, method of purification, choice of activity assay, reaction conditions (including choice of thiopurine substrate and reactant concentrations), use of units, and general presentation of results [24,28,29,30,31,32]. Both SAH and 6-Me MP coproducts inhibit the enzyme, albeit through different mechanisms; SAH is a potent competitive inhibitor with a K i of 0.75 µm with regard to SAM, and 6-Me MP is a non-competitive inhibitor with a K i of 560 µm with regard to 6-MP [10].…”

Individualized Therapy: Role of Thiopurine S-Methyltransferase Protein and Genetic Variants

“…A chaperone proteindependent, pro teasome-mediated pathway including aggresome formation and protein misfolding (30,31) and auto phagy (32), have been shown to contribute to degra dation of TPMT variant allozymes (12). An alteration at the active site of the enzyme also accounts for the loss-of-function of the TPMT enzyme (33). Altered pro tein quantity, as a consequence of TPMT protein degra dation, is an important mechanism responsible for the functional effects of an inherited variation in the amino acid sequence.…”