Deoxyuridylate phosphorylation

Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2 -deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of... 

Sugino el al. 17) have partly purified a deoxycytidylate kinase from calf thymus this preparation also phosphorylates cytidylate and uridylate, but not deoxyuridylate. The deoxyuridylate kinase activity was recovered in another protein fraction and so represents a distinct entity. As phosphate donors, only ATP and dATP were active in the cytidylate reaction. 

Thus, while existence of a cytidylate-deoxycytidylate kinase is established, there remains some uncertainty about the association of uridylate kinase activity with this enzyme. Phosphorylation of deoxyuridylate appears to be catalyzed by a separate enzyme activity, possibly by thymidylate kinase. 

Nelson and Carter 18) have purified thymidylate kinase about 5,000-fold from E. coli B and have shown that thymidylate was phosphorylated about seven times faster than deoxyuridylate. Uridylate was not a substrate, nor were adenylate, guanylate, cytidylate, or their deoxyribosyl counterparts. This enzyme was less specific for the triphosphate phosphoryl donor than the kinases previously discussed, because CTP, GTP, and the corresponding deoxyribonucleotides also served as phosphate donors, although less weU than ATP. 

Metabolism of the pyrimidine deoxyribonucleotides is more complex because, in addition to transfer of phosphoryl groups, deamination and methylation reactions occur at this level. Specifically, the thymidine phosphates are derived by methylation of deoxyuridylate, and the latter may be derived from the deoxycytidine phosphates by way of deoxycytidylate deaminase. The deoxycytidine phosphates are not formed by amination of deoxyuridine phosphates, but are derived entirely from the cytidine phosphates by enzymatic reduction (Chapter 16). 

Fractionation of extracts from calf thymus for kinase activity toward deoxyribonucleoside monophosphates has revealed the presence of at least four separate enzymes the kinase activities for dAMP, dGMP, dCMP, and dTMP are separate entities in this tissue 31). Each has been partly purified and examination of substrate specificities showed that the kinases for deoxyadenylate and deoxyguanylate also phosphorylate the ribosyl homologues, adenylate and guanylate, respectively. The deoxycytidylate kinase accepts as substrates both cytidylate and uridylate, but will not phosphor> late deoxyuridylate. The calf thymus thymidine monophosphate... 

Both the pyrimidines and the purines are built up from small precursor molecules which are readily available in the metabolic pool (page 185). The free bases are not synthesized as such but, while being assembled, the partially constructed ring structure reacts with a special phosphorylated pentose known as PRPP (5-phosphoribosyl-l-pyrophosphate) and forms a ribonucleotide. The deoxyribonucleotides, with the exception of TMP which is formed by methylation of deoxyuridylate, are formed by reduction of the corresponding ribonucleoside diphosphate. The conversion is precisely controlled by allosteric effects which ensure that all four deoxyribonucleotides are available in amounts appropriate for nucleic acid synthesis. 

Analogs of the natural deoxynucleoside triphosphates can be used for DNA synthesis, both in vivo and in vitro, but substitution of unnatural for natural deoxynucleotide must conform with the requirements for base-pairing in the Watson-Crick model of DNA. Thus with the purified enzyme system, thymine could be replaced by uracil or 5-bromouracil, 5-methyl- and 5-bromocytosine for cytosine, and hypoxanthine for guanine ( 6). Although chemically synthesized deoxyuridine triphosphate can be incorporated into DNA, there is apparently no kinase in nature which phosphorylates deoxyuridylate to the triphosphate stage. This may account for the absence of uracil nucleotides in DNA ( 6). 

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