2 -Deoxyribonucleoside 5 -phosphates

The ribonucleotide reductase system, consisting of ribonucleotide reductase, thioredoxin and thioredoxin reductase, catalyzes the irreversible reduction of the four common ribonucleoside-5 -phosphates to the corresponding 2 -deoxyribonucleoside-5 -phosphates. This reaction provides the deoxy ribonucleotide precursors necessary for DNA synthesis. 

In the approach using bis(tetrazolyl)morpholinophosphine for synthesizing bis (deoxyribonucleoside) phosphates it is not necessary to protect the 3 -hydroxyl group of die second deoxyribonucleoside (selective attack on the 5 -OH).[983,[1003 In this reaction bis(tetrazolyl)morpholinophosphine is superior to other bis(azolyl)moipholinophosphines or azolylchloromorpholinophosphines.11 13... 

RGURE 8-39 Nucleoside phosphates General structure of the nucleoside 5 -mono-, di-, and triphosphates (NMPs, NDRs, and NTRs) and their standard abbreviations In the deoxyribonucleoside phosphates (dNMPs, dNDPs, and dNTPs), the pentose is2 -deoxy-D-ribose. 

These enzymic reactions are essential to all living cells in that they provide the monomeric precursors for the de novo synthesis of DNA. The production of the 2 -deoxyribonucleoside phosphates required for DNA synthesis is carefully regulated through allosteric control of the enzyme by 2 -deoxynucleoside triphosphates and ATP, which regulate both overall activity and substrate specificity. 

As we have already noted, the deoxyribonucleoside phosphates occur in animal tissues in small amounts and their concentrations are increased in cells engaged in DNA synthesis. The triphosphates of the four deoxyri-bonucleosides represented in DNA have been demonstrated in cell extracts, as have the mono- and diphosphates of thymidine and deoxycytidine. The mono-, di-, and triphosphates of deoxyuridine are known as intermediary compounds in the metabolism of the pyrimidine deoxyribonucleotides. 

The following diagram illustrates in very broad terms routes by which cellular deoxyribonucleoside phosphates are derived and utilized, and is intended to provide perspective for the more detailed discussion of Chapters 14-16. 

A nucleoside consists of a purine or pyrimidine base linked to a pentose, either D-ribose to form a ribonucleo-side or 2-deoxy-D-ribose to form a deoxyribonucleoside. Three major purine bases and their corresponding ribo-nucleosides are adenine/adenosine, guanine/guanosine and hypoxanthine/inosine. The three major pyrimidines and their corresponding ribonucleosides are cytosine/ cytodine, uracil/uradine and thymine/thymidine. A nucleotide such as ATP (Fig. 17-1) is a phosphate or polyphosphate ester of a nucleoside. 

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)... 

DEOXYRIBONUCLEASES 2 -Deoxyribonucleoside diphosphate, RIBONUCLEOTIDE REDUCTASE 2-DEOXYRIBOSE-5-PHOSPHATE ALDOLASE DEOXYTHYMIDINE KINASE DEP,... 

The reaction catalyzed by polynucleotide phosphorylase differs fundamentally from the polymerase activities discussed so far in that it is not template-dependent. The enzyme uses the 5 -diphosphates of ribonucleosides as substrates and cannot act on the homologous 5 -triphos-phates or on deoxyribonucleoside 5 -diphosphates. The RNA polymer formed by polynucleotide phosphorylase contains the usual 3, 5 -phosphodiester linkages, which can be hydrolyzed by ribonuclease. The reaction is readily reversible and can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration. The probable function of this enzyme in the cell is the degradation of mRNAs to nucleoside diphosphates. 

Hog spleen acid DNase, as obtained by the above procedure, is completely free of contaminating phosphatase, exonuclease, and adenosine deaminase activities. The enzyme has a weak intrinsic hydrolytic activity on bis(p-nitrophenyl) phosphate and the p-nitrophenyl derivatives of deoxyribonucleoside 3 -phosphates (see Section III,D,3). 

Nucleotidase present in 48,000 X Q supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (7-4). 5 -UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75). This enzyme hydrolyzes all ribo-and deoxyribonucleoside 5 -phosphates (except dCMP and dTMP) with preference for 5 -IMP and 5 -UMP. The enzyme is strongly activated by Mn2+ Mg2+ is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (88, 86). 5 -Nucleotidase in mammary gland hydrolyzes all 5 -ribonucleotides and shows a decrease from pregnancy to early lactation (76). Rats injected with glucagon show increased 5 -nucleotidase in pancreatic islet tissue (77). The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isozymes (75). Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79). 

The following classes of phosphorus-containing compounds were not affected by large amounts of the enzyme with Mg2+, Mn2+, Zn2+, or Co2+ at either pH 9.1 or 7.5 (1) ribo- or deoxyribonucleoside mono-, di-, or triphosphates (2) ribo- or deoxyribopolynucleotides (3) nucleotide coenzymes (e.g., DPN+, UDP-glucose) (4) phosphomonoesters (e.g., glucose-6-P, p-nitrophenyl phosphate) (5) cyclic tri- or tetrametaphos-phates (6) phosphorofluoridates (inorganic phosphorofluoridate, adenosine 5 -phosphorofloridate) and (7) phosphonates (e.g., methylene-bis-phosphonate) (12, 57). 

K. Krawiec, B. Kierdaszuk and D. Shugar (2003). Inorganic tripolyphosphate (PPP ) as a phosphate donor for human deoxyribonucleoside kinases. Biochem. Biophys. Res. Commun., 301, 192-197. 

These four bases are incorporated into deoxyribonucleosides and deoxyribonucleotides similar to the bases in ribonucleosides and ribonucleotides. The following structures show the common nucleosides that make up DNA. The corresponding nucleotides are simply the same structures with phosphate groups at the 5 positions. 

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