Abstract

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines and thiopyrimidines. Human kidney TPMT was purified over 300-fold and its biochemical properties were determined. TPMT was "soluble" and had a molecular weight of approximately 36,000 daltons as estimated by gel filtration chromatography. The pH optimum of the purified TPMT was 6.7. "True" Km values for 6-mercaptopurine (6-MP) and S-adenosyl-L-methionine (SAM), the two cosubstrates for the reaction, were 0.30 mM and 2.7 microM respectively. "Apparent" Km values for 6-thioguanine and 2-thiouracil, two other methyl acceptor substrates, were 0.55 and 2.0 mM respectively. Aliphatic thiol compounds were either poor substrates for TPMT or were not methylated. S-Adenosyl-L-homocysteine was a competitive inhibitor of TPMT when the varied substrate was SAM, and 6-methylmercaptopurine was a noncompetitive inhibitor with respect to 6-MP. Purified TPMT was neither activated nor inhibited by 1 mM Ca2+ or Mg2+, but exposure to reagents such as N-ethylmaleimide and ethacrynic acid that interact with sulfhydryl groups inactivated the enzyme. Tropolone inhibited TPMT with a Ki of approximately 0.85 mM. Finally, human kidney TPMT activity could be distinguished from human kidney thiol methyltransferase (EC 2.1.1.9) activity on the basis of subcellular distribution, substrate specificity, kinetic characteristics and differential sensitivity to inhibitors.