Deoxyribonucleoside monophosphate phosphorylation kinases

Resolution of the enzymatic steps involved in converting each particular deoxyribonucleoside monophosphate to its di- and triphosphate derivatives has been accomplished through isolation and purification of the participating enzymes. It has turned out that in the deoxy series, 3- and y-phos-phates are added in the same way as in the ribo series in fact, it appears that most of the transphosphorylations are accomplished by kinases which will accept either the ribosyl or deoxyribosyl version of their particular substrates. Thus, the three levels of phosphorylation in the deoxyribonucleotide pool are interconnected by freely reversible transphosphorylation reactions (see also Chapter 4). 

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... 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

Another salvage route to making deoxyribonucleotides is via deoxyribonucleoside kinases, which can phosphorylate nucleosides to make nucleoside monophosphates. Human cells have four different deoxyribonucleoside kinases ... 

The kinase which converts deoxycytidine to its 5 -monophosphate has been studied most extensively in preparations from calf thymus 35, 36). The preferred substrate is deoxycytidine, for which the Michaelis constant (5 X 10 M) is much lower than that of two other substrates, deoxyadenosine and deoxyguanosine. Cytidine, uridine, and thymidine are not phosphorylated by this enzyme. Deoxycytidine kinase is subject to a complex pattern of allosteric regulation by nucleotides. The end product of deoxycytidine phosphorylation, dCTP, is a potent inhibitor this inhibition is reversed by dTTP. The enzyme has a rather broad specificity for the phosphate donor, with the triphosphates of the natural ribo- and deoxyribonucleosides being substrates the inactivity of dCTP is a notable exception. 

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