Nucleoside triphosphates, synthesis

Shankar, S. Hershberger, C.D. Chakrabarty, A.M. The nucleoside diphosphate kinase of Mycobacterium smegmatis identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme. Mol. Microbiol., 24, 477-487 (1997)... 

Fig. 13. Dependence on ATP and ITP concentration of calcium dependent phosphate liberation (a) and the corresponding nucleoside triphosphate synthesis on the concentration of ADP, IDP and GDP. 13 a ATP, ITP, 13b ADP, IDP, GDP...

Scheme 37 Second and third stages of the evolution of amino acid activation (2) The system of Scheme 39 is improved by enzyme catalysis the mixed anhydride is stabilized by binding making the equilibrium with NCA more favorable (3) an alternative pathway for nucleoside triphosphate synthesis has been introduced, the chemical flux has been reverted...

Fig. 9.7 31P NMR spectra obtained from a nucleoside triphosphate synthesis using phosphorus(V) chemistry, (a) Before MPLC/HPLC (b) After MPLC/HPLC.

Use Enzyme cross-linking agent, condensing agent for nucleoside triphosphate synthesis. 

P. Brazdilova, M. Vrabel, R. Pohl, H. Pivonkova, L. Havran, M. Hocek, and M. Fojta, Ferrocenylethynyl derivatives of nucleoside triphosphates Synthesis, incorporation, electrochemistry, and bioanalytical applications, Chemistry 13[34], 9527-9533 [2007]. 

The net free energy change, AG°, for this conversion is —37.7 kj/mol. The consumption of a total of six nucleoside triphosphates drives this process forward. If glycolysis were merely reversed to achieve the net synthesis of glucose from pyruvate, the net reaction would be... 

Fontes, R., Dukhovich, A., Sillero, A., and Gunther Sillero, M. A. (1997). Synthesis of dehydroluciferin by firefly luciferase, effect of dehydrolu-ciferin, coenzyme A and nucleoside triphosphates on the luminescence reaction. Biochem. Biophys. Res. Commun. 237 445—450. 

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). 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

As already mentioned, a continual inflow of energy is necessary to maintain the stationary state of a living system. It is mostly chemical energy which is injected into the system, for example by activated amino acids in protein biosynthesis (see Sect. 5.3) or by nucleoside triphosphates in nucleic acid synthesis. Energy flow is always accompanied by entropy production (dS/dt), which is composed of two contributions ... 

Nucleotides are needed for DNA and RNA synthesis (DNA replication and transcription) and for energy transfer. Nucleoside triphosphates (ATP and GTP) provide energy for reactions that would otherwise be extremely unfevorable in the cell. 

Figure 4.15 k summary of the fate of nucleosides that are produced from RNA digestion in the lumen of the intestine. The nucleosides produced from RNA in the lumen are absorbed by the enterocytes and then transported from the intestine into the blood from where they are taken up by cells (especially proliferating cells, e.g. in the bone marrow) to form nucleotides for DNA and RNA synthesis. (See Chapter 10) NTP is nucleoside triphosphate. 

Each new double helix is comprised of one strand that was part of the original molecule and one strand that is newly synthesized. Not surprisingly, this is a very simplistic description of a quite complex process, catalysed by enzymes known as DNA polymerases. The precursors for synthesis of the new chain are the nucleoside triphosphates, dATP, dGTP, dTTP, and dCTP. We have already met ATP when we considered anhydrides of phosphoric acid (see Box 7.25) these compounds are analogues of ATP, though the sugar is deoxyribose rather than ribose. 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

E. Trevisiol, E. Defrancq, J. Lhomme, A. Laayoun, P. Cros, Synthesis of nucleoside triphosphates that contain an aminooxy for post-amplification labelling, Eur. J. Org. Chem. (2000) 211-217. 

FIGURE 13-11 Nucleoside triphosphates in RNA synthesis With each nucleoside monophosphate added to the growing chain, one PPi is released and hydrolyzed to two P,. The hydrolysis of two phosphoanhydride bonds for each nucleotide added provides the energy for forming the bonds in the RNA polymer and for assembling a specific sequence of nucleotides... 

MECHANISM FIGURE 26-1 Transcription by RNA polymerase in E. coli. For synthesis of an RNA strand complementary to one of two DNA strands in a double helix, the DNA is transiently unwound, (a) About 17 bp are unwound at any given time. RNA polymerase and the bound transcription bubble move from left to right along the DNA as shown facilitating RNA synthesis. The DNA is unwound ahead and rewound behind as RNA is transcribed. Red arrows show the direction in which the DNA must rotate to permit this process. As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand extruded. The RNA polymerase is in close contact with the DNA ahead of the transcription bubble, as well as with the separated DNA strands and the RNA within and immediately behind the bubble. A channel in the protein funnels new nucleoside triphosphates (NTPs) to the polymerase active site. The polymerase footprint encompasses about 35 bp of DNA during elongation. 

Substrate specificity site The binding of nucleoside triphosphates to an additional allosteric site (known as the substrate specificity site) on the enzyme regulates substrate specificity, causing an increase in the conversion of different species of ribonucleotides to deoxyribonucleotides as they are required for DNA synthesis. 

Several chemical and enzymatic methods are available for the synthesis of glycosyl phosphates. The required nucleoside triphosphates (NTPs) are most conveniently prepared by enzymatic routes. In general, these methods involve the sequential use of two kinases to transform NMPs to NTPs, by the corresponding NDPs (Fig. 2, see a recent review [12] for more details). 

Figure 2 Synthesis of nucleoside triphosphates (1) Adenylate kinase (EC 2.1 A3, N = A,C,U) or guanylate kinase (EC 2.7.4.8, N = G), or nucleoside monophosphate kinase (EC 2.7.4.4, N = U). (2) Pyruvate kinase (EC 2.7.1.40). NMP, nucleoside monophosphate NDP, nucleoside diphosphate NTP, nucleoside triphosphate. (From Ref. 12.)...

Before the sequencing begins it is necessary to prepare a short primer that is complementary to a sequence at one end of the DNA strand to be sequenced. This may be prepared enzymatically,622 623 or by non-enzymatic synthesis. The short primer is annealed to the end of the DNA and the resulting molecule is incubated with a DNA polymerase and a mixture of the four mononucleotide triphosphates, one of which is radio-labeled in this position. Four reaction mixtures are prepared. Each mixture contains all four nucleoside triphosphates and also one of four different chain-terminating inhibitors, the most popular of which are the 2, 3 -dideoxyribonucleoside triphosphates ... 

Ribonucleotide reductases are discussed in Chapter 16. Some are iron-tyrosinate enzymes while others depend upon vitamin B12, and reduction is at the nucleoside triphosphate level. Mammalian ribonucleotide reductase, which may be similar to that of E. coli, is regarded as an appropriate target for anticancer drugs. The enzyme is regulated by a complex set of feedback mechanisms, which apparently ensure that DNA precursors are synthesized only in amounts needed for DNA synthesis.273 Because an excess of one deoxyribonucleotide can inhibit reduction of all... 

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