Nucleoside 5’-triphosphates pyrimidine

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). 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized... 

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

This enzyme is notable in that it is not specific for the base (purines or pyrimidines) or the sugar (ribose or deoxyribose). This nonspecificity applies to both phosphate acceptor (A) and donor (D), although the donor (NTPD) is almost invariably ATP, because it is present in higher concentration than other nucleoside triphosphates under aerobic conditions. 

Restriction enzymes which cleave double-stranded DNA, making staggered cuts which leave the 5 -end of each strand extended. Double-stranded DNA with protruding 5 -ends is more efficiently phosphorylated by polynucleotide kinase than DNA with flush or recessed 5 -ends. The fore-shortened 3 -ends can also be labelled by extending them with 32P-labelled nucleoside triphosphates complementary to bases in the extended template strand using DNA polymerase. Y—pyrimidine nucleotide, R—purine nucleotide, N—any nucleotide. A complete list of commercially obtainable restriction endonucleases is given in Appendix III. 

How is the other major pyrimidine ribonucleotide, cytidine, formed It is synthesized from the uracil base of UMP, but UMP is converted into UTP before the synthesis can take place. Recall that the diphosphates and triphosphates are the active forms of nucleotides in biosynthesis and energy conversions. Nucleoside monophosphates are converted into nucleoside triphosphates in stages. First, nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor (Section 9.4). For example, UMP is phosphorylated to UDP by UMP kinase. 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Novel 2 -modified nucleoside triphosphate derivatives incorporating imidazole, amino or carboxylate pendant groups attached to the 5-position of pyrimidine base through alkynyl and alkyl spacers (115) have been synthesised. In one reported procedure, the appropriately protected modified nucleoside was phosphorylated with POCI3 in triethylphosphate in the presence of 1,8-bis(dimethylamino)naphthalene. The phosphorochloridate intermediate was then condensed in situ with tri-n-butylammonium pyrophosphate to yield the protected triphosphate analogue that was then deprotected. In another... 

The situation becomes curiouser and curiouser when the ATGase reaction takes place not in the presence of GTP, a pyrimidine nucleoside triphosphate, but in the presence of adenosine triphosphate (ATP), a purine nucleoside triphosphate. The structural similarities between GTP and ATP are apparent, but ATP is not a product of the pathway that includes the reaction of ATGase and that produces GTP. Both ATP and GTP are needed for the synthesis of RNA and DNA. The relative proportions of ATP and GTP are specified by the needs of the organism. If there is not enough GTP relative to the amount of ATP, the enzyme requires a signal to produce more. In the presence of ATP, the rate of... 

ATP is the primary source of utilizable energy for biosynthetic reactions, but other purine and pyrimidine nucleoside triphosphates (GTP, UTP, CTP) occur in cells and are formed by phosphoryl transfer from ATP, as described in detail in Chapter 4. These are also high-energy compounds, and their hydrolysis can be coupled to energetically unfavorable reactions. This most commonly takes place through nucleotidyl transfer reactions leading to group-transfer coenzymes (Section II, B, 3). 

In the absence of Mg + the two purine nucleoside triphosphates compete for a single binding site, one presumably involved with initiation. With Mg +, they compete for a second site, and the two pyrimidine nucleoside triphosphates also compete for a single site. The Michaelis constants for the nucleotide substrates are in the range 0.05 to 0.3 nM, but vary with the DNA template. 

The nucleoside diphosphate kinase from human erythrocytes was of low specificity and would react with nucleoside triphosphates and nucleoside diphosphates which contained either ribose or deoxyribose and any of the natural purine or pyrimidine bases. The finding that alternative substrates, such as GTP and ATP, were competitive inhibitors, is consistent with the low substrate specificity... 

The ability of nucleoside triphosphates, singly, or in combination, to effect changes in the catalytic properties of the reductase have been explored systematically by Reichard and his co-workers the complicated pattern of results is summarized in Table 16-11. These findings have been interpreted in terms of four states of activity for the reductase. In the presence of ATP, the reductase prefers as substrates pyrimidine nucleoside diphosphates. In the presence of dGTP, a purine-specific state is assumed and dTTP causes the enzyme to be in a condition in which both purine and pyrimidine ribonucleoside diphosphates are reduced. 

Figure 28 Pyrimidine nucleoside triphosphate monomers enzymatically installed by PCR, which are then converted into aldehyde nucleation sites for sUvot deposition by reacting with Tollens solution.

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