Other Nucleotide Phosphates

Magnesium is the fourth most abundant cation in the body after sodium, potassium, and calcium, and is the second most abundant cation in intracellular fluid after K+ Mg + is needed in many enzymatic reactions, particularly those in which ATP Mg + is a substrate. Magnesium binds to other nucleotide phosphates and to nucleic acids and is required for DNA replication, transcription, and translation. The DNA helix is stabilized by binding... 

Fig. 5.10. The formula of one of the mononuclear molybdenum cofactors, Moco. Others have a nucleotide phosphate extension (see references to these elements in Further Reading). In sulfide-rich environments, tungsten replaced molybdenum. In some coenzymes, two pterins are bound to the metal ions.

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)... 

This enzyme [EC 2.7.1.40] catalyzes the reaction of ADP with phosphoenolypyruvate to produce ATP and pyruvate. Other nucleotides that can be used as substrates include UDP, GDP, CDP, IDP, and dADP. The enzyme will also phosphorylate hydroxylamine and fluoride in the presence of carbon dioxide. See Nucleoside 5 -Tri-phosphate Regeneration... 

The sapphyrin-modified silica gels also proved effective for HPLC separation of various anions other than phosphates, but not neutral or cationic species. For instance, various monoanionic species such as diphenyl phosphate, benzene sulfonic acid, phenyl arsenate, phenyl phosphate, and benzoate could all be separated from each other. Nucleotide mono-, di-, and triphosphates such as AMP, ADP, and ATP were also retained on these columns and were found to be readily separable from one another under isochratic HPLC conditions. Likewise, in what is still unpublished work, it has been found that these columns can be used to separate... 

ATP is the primary high-energy phosphate compound produced by catabolism, in the processes of glycolysis, oxidative phosphorylation, and, in photosynthetic cells, photophosphorylation. Several enzymes then cany phosphoryl groups from ATP to the other nucleotides. Nucleoside diphosphate kinase, found in all cells, catalyzes the reaction... 

Ribonuclease T2 is regarded as a nonspecific endoribonuclease [EC 2.7.7.17, ribonucleate nucleotido-2 -transferase (cyclizing)]. It preferentially splits the internucleotide bonds between the 3 -adenylic acid group and the 5 -hydroxyl group of adjacent nucleotides in RNA, with the intermediary formation of adenosine 2, 3 -cyclic phosphate and splits consequently all secondary phosphate ester bonds of other nucleotides in RNA via the nucleotides 2, 3 -cyclic phopshates. 

It was found that the polymer exhibited selectivity towards phosphomonoester dianions. Less polar compounds were found to bind non-specifically to the polymer. The polymer was then used as a stationary phase for a HPLC column. A mixture containing dA, 5 -dAMP and 3, 5 -cAMP was thus separated. As expected, the retention time of 5 -AMP was larger than those for dA and 3, 5 -cAMP. The same was tme for other nucleotides compared to the corresponding nucleosides. When the Zn2+-free control polymer was used, all compounds were immediately eluted. The possibility to use polymer-anchored recognition units to separate biologically important phosphates was thus proved. 

A tetraamidinium functionalized, bowl-type cavitand (receptor 8) was developed by Diederich and Sebo [47]. This receptor was found to complex 1,3-dicarboxylate anions with good selectivity and a 1 2 binding stoichiometry both in CD3OD and D20, as revealed by standard Job plot analysis. In contrast, various nucleotide phosphates were found to be bound with a 1 1 stoichiometry in D20. In the case of the adenosine phosphates, the association constants increased as a function of nucleotide charge [i.e., the affinity order (K., M-1) was cAMP (1400) < AMP (10000)charged receptor (8) also showed moderate selectivity towards AMP (Ka = 10 000 M-1) over other nucleotide monophosphate anions, such as GMP (FCa = 5200 M-1), CMP (fCa = 3500 M-1), TMP (K l — 5900 M ), and UMP (Ka-3800M ) in D20 containing TRIS buffer (2.5 mM, pH 8.3). 

Finally, we point out that CT from the sugar-phosphate (SP) unit to the cytosine (C) molecule cannot be explained by the naive HOMO (D)-LEMO (A) picture, because the valence band of polySP (see Table 10) lies below the valence band of polyC by about 0.8 eV. The same is true if one compares the upper limits of the valence bands of the three other nucleotide base stacks (see Table 9) with the upper limits of the valence band of polySP. 

NAD was separated from other nucleotides by chromatography on a Waters Partisil 10-SAX column (8 mm x 100 mm). The column was equilibrated with 5 mM ammonium phosphate buffer (pH 2.9). After injection, the same solvent was used for 10 minutes at a flow rate of 1 mL/min, and then for 2 minutes at 1.5 mL/min. A mobile phase was then switched to 0.65 M ammonium phosphate (pH 3.7) for 8 minutes at a flow rate of 2 mL/min, after which the column was reequilibrated with the initial solvent. Fractions from the NAD area were collected and counted by scintillation spectrometry. 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

Begirming and ending with ornithine, the reactions of the cycle consumes 3 equivalents of ATP and a total of 4 high-energy nucleotide phosphates. Urea is the only new compound generated by the cycle all other intermediates and reactants are recycled. 

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