METHYLATION OF MERCAPTOPURINE, THIOGUANINE, AND THEIR NUCLEOTIDE METABOLITES BY HETEROLOGOUSLY EXPRESSED HUMAN THIOPURINE S-METHYLTRANSFERASE

Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibit s genetic polymorphism, catalyzes S-methylation of mercaptopurine (MP) and thioguanine (TG), yielding S-methylated nucleobases that are inac tive, whereas S-methylated nucleotides of these thiopurines are cytoto xic. A yeast-based heterologous expression system was therefore used t o characterize human TPMT-catalyzed methylation of MP, TG, and their p rincipal nucleotide metabolites [thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively]. MP, TG, TIMP, and T GMP were all substrates for human TPMT, exhibiting similar Michaelis-M enten kinetic parameters (K-m, 10.6-27.1 mu M; V-max, 31-59 nmol/min/m g of TPMT). Consistent with these kinetic parameters, human leukemia c ells (CEM) incubated for 24 hr with 10 mu M MP or TG accumulated signi ficantly higher (2.3-fold, p = 0.01) concentrations of methyl-TIMP aft er MP incubation than methyl-TGMP after TG incubation, due to the 2.7- fold higher concentration of TIMP after MP incubation, compared with T G nucleotides (TGN) after TG incubation. Moreover, intracellular accum ulation of TGN was 2.5-fold greater after TG incubation than after MP incubation (p = 0.01). These data establish that MP, TG, and their pri ncipal nucleotide metabolites are comparable substrates for polymorphi c TPMT, and they demonstrate significant differences in the accumulati on of active TGN and methylated nucleotides when leukemia cells are tr eated with MP versus TG.