Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.1.1.67 (thiopurine methyltransferase)
 551 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An assay is described for the determination of red blood cell (RBC) thiopurine methyltransferase (TPMT) activity. TPMT S-methylates the antileukaemic drugs 6-mercaptopurine (6-MP) and 6-thioguanine and enzyme activity is inherited as a genetic trait. The assay is based on the TPMT-catalysed conversion of 6-MP to 6-methylmercaptopurine (methyl-MP) with non-radioactive S-adenosyl-L-methionine as the methyl donor. The methyl-MP metabolite is extracted into toluene as a phenyl-mercury adduct and back-extracted into 0.1 M HCl. Methyl-MP is quantitated by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet detection using a C18 Resolve cartridge and a mobile phase of methanol-water (20:80, v/v) with 100 mM triethylamine adjusted to pH 3.2 with orthophosphoric acid. There was a strong correlation between the HPLC assay and the established radiochemical assay (P < 0.0001). TPMT activities were measured by both methods in a population study of 111 children. There was no significant difference between the two frequency distribution histograms (P > 0.6).
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PMID:High-performance liquid chromatographic assay of human red blood cell thiopurine methyltransferase activity. 786 49

Human thiopurine S-methyltransferase (TPMT) is an essential protein in 6-mercaptopurine (6MP) drug metabolism. To understand the pharmacogenetics of TPMT and 6MP, X-ray co-crystal structures of TPMT complexes with S-adenosyl-L-methionine (AdoMet) and 6MP are required. However, the co-crystal structure of this complex has not been reported because 6MP is poorly water soluble. We used molecular dynamics (MD) simulation to predict the structure of the complex of human TPMT-AdoHcy(CH(2))6MP, where the sulfur atoms of AdoHcy and 6MP were linked by a CH(2) group. After 1300 picoseconds of MD simulation, the trajectory showed that 6MP was stabilized in the TPMT active site by formation of non-bonded interactions between 6MP and Phe40, Pro196 and Arg226 side chains of TPMT. The intersulfur distance between AdoHcy and 6MP as well as the binding modes and the interactions of our TPMT-AdoHcy model are consistent with those observed in the X-ray crystal structure of murine TPMT-AdoHcy-6MP complex. The predicted binding modes of AdoHcy and 6MP in our model are consistent with those observed in murine TPMT X-ray crystal structures, which provides structural insights into the interactions of TPMT, AdoHcy, and 6MP at the atomic level and may be used as a starting point for further study of thiopurine drug pharmacogenetics.
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PMID:Molecular dynamics simulation of a human thiopurine S-methyltransferase complexed with 6-mercaptopurine model. 2019 69

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/mol, which is in good agreement with the available experimental rate constant data.
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PMID:Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations of the thiopurine S-methyltransferase reaction with 6-mercaptopurine. 2161 36