Deoxyribonucleotide kinases

Streptococcus (Dipiococcus) pneumoniae DNA-membrane-complex preparation gel electrophoresis DNA, RNA, protein, phospholipids, ribonucleotide reductase, deoxyribonucleotide kinases, DNA ligase, nucleases, DNA polymerase 337, 338... 

Adenosine deaminase deficiency results in an accumulation of adenosine, which is converted to its ribonucleotide or deoxyribonucleotide forms by cellular kinases. As dATP levels rise, they inhibit ribonucleotide reductase, thus preventing the production of deoxyribonucleotides, so that the cell cannot produce DNA and divide. This causes severe combined immunodeficiency disease, involving a lack of T cells and B cells. 

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 activity of mitochondrial thymidine kinase is sufficiently broad that it will also act on the anti-HIV drug, 3 -azido-2 3 -dideoxythymidine (AZT). That is, the enzyme can phosphorylate AZT to a deoxyribonucleotide of azidothymidine, which is then incorporated into DNA. Evidence suggests that deoxyribonucleotides of AZT interfere with mitochondrial function, possibly by inhibiting mitochondrial DNA replication or transcription, which may explain some of the side effects of cardiotoxicity (damage to the heart muscle) observed with its use. 

See also Nucleotide Analogs in Selection, Deoxycytidine Kinase, BrdUrd, Salvage Routes to Deoxyribonucleotide Synthesis, Nucleotide Analogs in Medicine... 

Purine deoxyribonucleotides are derived primarily from the respective ribonucleotide (Fig. 6.2). Intracellular concentrations of deoxyribonucleotides are very low compared to ribonucleotides usually about 1% that of ribonucleotides. Synthesis of deoxyribonucleotides is by enzymatic reduction of ribonucleotide-diphosphates by ribonucleotide reductase. One enzyme catalyzes the conversion of both purine and pyrimidine ribonucleotides and is subject to a complex control mechanism in which an excess of one deoxyribonucleotide compound inhibits the reduction of other ribonucleotides. Whereas the levels of the other enzymes involved with purine and pyrimidine metabolism remain relatively constant through the cell cycle, ribonucleotide reductase level changes with the cell cycle. The concentration of ribonucleotide reductase is very low in the cell except during S-phase when DNA is synthesized. While enzymatic pathways, such as kinases, exist for the salvage of pre-existing deoxyribosyl compounds, nearly all cells depend on the reduction of ribonucleotides for their deoxyribonucleotide... 

In the absence of ADA activity, both adenosine and deoxyadenosine will accumulate. When deoxyadenosine accumulates, adenosine kinase can convert it to dAMP. Other kinases will allow dATP to then accumulate within the lymphocyte. Why specifically the lymphocyte The other cells of the body are secreting the deoxyadenosine they cannot use, and it is accumulating in the circulation. As the lymphocytes are present in the circulation, they tend to accumulate this compound more so than cells not constantly present within the blood. As dATP accumulates, ribonucleotide reductase becomes inhibited, and the cells can no longer produce deoxyribonucleotides for DNA synthesis. Thus, when cells are supposed to grow and differentiate in response to cytokines, they cannot, and they die. 

S Additional information <3, 7> (<3> enzyme exists in different conformational states with different substrate kinetic properties [9] <3> presumably one common nucleoside acceptor site [15] <3> purine deoxynucleo-side activity inseparably associated with deoxycytidine kinase protein [16] <3> several isozymes cytosolic deoxycytidine kinase I and II, plus mitochondrial isozyme [10] <3> multisubstrate enzyme, that also phos-phorylates purine deoxyribonucleotides [9] <7> enzyme has two separate active sites for deoxycytidine and deoxyadenosine activity [22] <3> reacts with both enantiomers of -deoxycytidine, -deoxyguanidine, -deoxyadenosine, and a-D-deoxycytidine is also substrate [31] <3> reacts with both enantiomers of jS-deoxyadenosine, j3-arabinofuranosyl-adenine and jd-deoxyguanine ]34] <3> remarkably relaxed enantioselectivity with respect to cytidine derivatives in p configuration [36] <3> lack of enantioselectivity for D- and L-analogues of cytidine and adenosine [43]) [9, 10, 15, 16, 22, 31, 34, 43]... 

The acceptability of deoxyguanylate and dATP as substrates in the guanylate kinase reaction is noteworthy there does not appear to be a particular group of nucleoside monophosphate kinases specific for deoxyribonucleotides (see Chapter 14), apart from that for thymidylate and deoxyuridylate. 

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. 

Although cells derive their deoxyribonucleotides primarily by reduction of ribonucleotides, the deoxyribonucleosides can be utilized to some extent by way of kinase reactions. Phosphorylation of deoxyribonucleosides represents the only known point of entry into the sequences of deoxyribonucleotide metabolism other than the main entry point, ribonucleotide reduction. Pyrimidine deoxyribonucleosides may be incorporated into DNA in animal cells by this route. The conversion of deoxyadenosine and deoxyguanosine into deoxyribonucleotides and thence into DNA would also appear possible by a kinase-initiated sequence, because the enzymatic phosphorylation of these compounds has been demonstrated. However, this route has not been well studied in animal cells and its assessment is complicated by very active deamination and phosphorolytic cleavage reactions which compete with the reactions leading to DNA. E. coli cells appear to possess only one kinase capable of phosphorylating deoxyribonucleosides, thymidine kinase. 

A means of converting free bases into deoxyribonucleotides would appear to be afforded by the sequential action of nucleoside phosphorylases and kinases however, cells have only a very limited capability for the endogenous formation of the deoxyribose 1-phosphate needed for this... 

The reversibihty of deoxyribonucleoside phosphorolysis suggests that bases might be elevated to the deoxyribonucleotide level by successive actions of a phosphorylase and a kinase ... 

An additional feature of thymidine kinase activity is that it is subject to allosteric regulation. Despite the implication in these facts that this enzyme activity is important in the deoxyribonucleotide economy of cells, thymidine is not an obligatory intermediate in the biosynthesis of the thymidine phosphates and the mutational loss of this enzyme is not lethal. 

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). 

Thymidine kinase serves as a salvage reaction in the phosphorylation of thymidine to yield dTMP. Its activity is under allosteric control as revealed by Okazaki and Kornberg with a highly purified preparation from E. coli [186]. Sigmoidal kinetics are obtained with ATP as substrate, and this is converted to a hyperbolic form by dCDP which functions as an activator. The affinity for thymidine is also increased by dCDP. Feedback inhibition is obtained with dTTP, the end product of the pathway. Inhibition by dTTP is competitive with the phosphate acceptor, thymidine, and noncompetitive with the donor, ATP. The kinetics of inhibition in the presence of the activator are difficult to interpret in terms of a simple competition between dTTP and dCDP for an allosteric site. This type of control apparently serves the same function as that described for dCMP deaminase above where the activity of the enzyme is decreased by the end product, dTTP, and increased when other deoxyribonucleotides accumulate. 

Thymidine must first be converted to its triphosphate (TTP) before incorporation as the deoxyribonucleotide (dTTP) into the DNA molecule. Thus two enzymes must be present for thymidine to be utilized (1) thymidine kinase (ATP thymidine 5 phosphotransferase), for phosphorylation of thymidine and (2) DNA polymerase, for incorporation of deoxyribonucleotides on the DNA template. Limiting levels of either of these enzymes in seeds could obviously restrict DNA synthesis and, as a corollary, DNA synthesis could be regulated by either of these enzymes. 

The mode of nthesis of deoxyribose is a subject of active interest at the present time. It has been shown that deoxyribose 5-phosphate can be synthesized by condensation of acetaldehyde and glyceraldehyde 3-phosphate in the presence of an aldolase from animals and bacteria (80). A pathway for deoxyribonucleotides can be formulated which employs this aldolase. The 5-phosphate ester could be converted to a l-phosphate by a mutase, the deoxypentose phosphate condensed with a base to form a nucleoside by nucleoside phosphorylase, and the nucleoside converted to a nucleotide by a kinase [Eq. (26)]. 

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