Kinetic evidence for covalent phosphoryl-enzyme intermediate in phosphotransferase activity of human red cell pyrimidine nucleotidases

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This chapter illustrates the way in which kinetic analysis provides convincing evidence for a covalent enzyme-substrate intermediate and also shows that the pyrimidine nucleotidases-I (PN-I) phosphotransferase activity displays higher affinity for oxynucleosides with respect to deoxynucleosides, whereas the contrary seems to be true for pyrimidine nucleotidases-II (PN-II). The essential feature of each intermediate species in the above reactions is that the intermediate must be of sufficient reactivity to be returned to a respective monoester species. Despite the fact that the putative phosphoryl-enzyme intermediate remains to be chemically characterized, the chapter presents evidence that human red cell pyrimidine nucleotidases operate by so-called “branched mechanisms,” with water and pyrimidine nucleosides competing as phosphoryl acceptors. Beyond the interesting catalytic features of PN-I and PN-II, the spectrum of substrates with which phosphotransfer activity can be observed may also prove to be invaluable in comprehending the action of antineoplastic agents and other unphosphorylated prodrugs. Although most cells presumably possess the ability to prevent the formation of undesirable metabolic by-products, the transfer reactions described in the chapter may represent opportunities for therapeutic intervention.