Nucleoside 5’-triphosphates

Closely related to ATP, and present in biological systems, are a number of other nucleoside triphosphates and their corresponding diphosphates and monophosphates. These are uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and thymidine triphosphate (TTP), all of which have about the same free energy of hydrolysis as ATP (11.29). In addition there are the deoxy nucleoside triphosphates (Chapter 10.4). 

Although not as universally important as ATP, GTP is involved in many essential body processes such as cellular signalling, protein synthesis and ion channel regulation. 

Important functions of the principal energy-carrying compounds can be summarised as follows  

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FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

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... 

Sugar nucleotides are formed from sugar-l-phosphates and nucleoside triphosphates by specific pyrophosphorylase enzymes (Figure 23.18). For example, UDP-glucose pyrophosphorylase catalyzes the formation of UDP-glucose from glucose-l-phosphate and uridine 5 -triphosphate ... 

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. 

Ligases catalyze the joining together of two molecules, coupled to the hydrolysis of a pyrophospho-ryl group in ATP or a similar nucleoside triphosphate. 

Other Nucleoside Triphosphates Participate in the Transfer of High-Energy Phosphate... 

Nucleoside Triphosphates Have High Group Transfer Potential... 

Nonhydrolyzable Nucleoside Triphosphate Analogs Serve as Research Tools... 

Synthetic nonhydrolyzable analogs of nucleoside triphosphates (Figure 33-13) allow investigators to distinguish the effects of nucleotides due to phosphoryl transfer from effects mediated by occupancy of allosteric nucleotide-binding sites on regulated enzymes. 

Nucleoside triphosphates have high group transfer potential and participate in covalent bond syntheses. The cyclic phosphodiesters cAMP and cGMP function as intracellular second messengers. 

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). 

Nick translation A technique for labeling DNA based on the ability of the DNA polymerase from E colt to degrade a strand of DNA that has been nicked and then to resynthesize the strand if a radioactive nucleoside triphosphate is employed, the rebuilt strand becomes labeled and can be used as a radioactive probe. 

Most nucleotide sugars are formed in the cytosol, generally from reactions involving the corresponding nucleoside triphosphate. GMP-sialic acids are formed in the nucleus. Formation of uridine diphosphate galactose (UDP-Gal) requires the following two reactions in mammahan tissues ... 

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

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. ... 

Nucleoside triphosphates are prepared analogously to the diphosphates by the methods a, b, c, and d, representing conversion of a nucleoside phosphoric azolide with inorganic pyrophosphate (Method a), of a nucleoside diphosphoric azolide with inorganic phosphate (Method b), of a phosphoric bisazolide with a nucleoside diphosphate (Method c), and of a diphosphoric bisazolide (bisazolide of pyrophosphoric acid) with a nucleoside phosphate (Method d). 

Other nucleoside triphosphates modified in the a- or / -positions were obtained according to the following routes ... 

Many of the mutations caused by artificially produced base analogues are transitions. Mutations are produced by base analogues in one of two different ways. On entering the cell, a base analogue is converted to a nucleoside triphosphate that base pairs, perhaps incorrectly, with a DNA template and is inserted into the nucleotide chain. This is one way in which the mutation can be produced. The other requires an additional round of replication so that an improper base pair forms as a result of the previously incorporated analogue. The result in both cases is a permanently modified DNA. 

The thioester hypothesis can be summed up as follows the formation of thiols was possible, for example, in volcanic environments (either above ground or submarine). Carboxylic acids and their derivatives were either formed in abiotic syntheses or arrived on Earth from outer space. The carboxylic acids reacted under favourable conditions with thiols (i.e., Fe redox processes due to the sun s influence, at optimal temperatures and pH values) to give energy-rich thioesters, from which polymers were formed these in turn (in part) formed membranes. Some of the thioesters then reacted with inorganic phosphate (Pi) to give diphosphate (PPi). Transphosphorylations led to various phosphate esters. AMP and other nucleoside monophosphates reacted with diphosphate to give the nucleoside triphosphates, and thus the RNA world (de Duve, 1998). In contrast to Gilbert s RNA world, the de Duve model represents an RNA world which was either supported by the thioester world, or even only made possible by it. 

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 ... 

The unique properties of oligonucleotides create crosslinking options that are far different from any other biological molecule. Nucleic acids are the only major class of macromolecule that can be specifically duplicated in vitro by enzymatic means. The addition of modified nucleoside triphosphates to an existing DNA strand by the action of polymerases or transferases allows addition of spacer arms or detection components at random or discrete sites along the chain. Alternatively, chemical methods that modify nucleotides at selected functional groups can be used to produce spacer arm derivatives or activated intermediates for subsequent coupling to other molecules. 

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