Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells.
Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.
D002460:Cell Line; D003844:DCMP Deaminase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009709:Nucleotide Deaminases / Q000378:metabolism*
D046911:Macromolecular Substances; D009709:Nucleotide Deaminases; D003844:DCMP Deaminase