Nucleoside triphosphate-magnesium

DNA polymerases catalyze DNA synthesis in a template-directed manner (Box 16). For most known DNA polymerases a short DNA strand hybridized to the template strand is required to serve as a primer for initiation of DNA synthesis. Nascent DNA synthesis is promoted by DNA polymerases by catalysis of nucleophilic attack of the 3 -hydroxyl group of the 3 -terminal nucleotide of the primer strand on the a-phosphate of an incoming nucleoside triphosphate (dNTP), leading to substitution of pyrophosphate. This phosphoryl transfer step is promoted by two magnesium ions that stabilize a pentacoordinated transition state by complex-ation of the phosphate groups and essential carboxylate moieties in the active site (Figure 4.1.1) [2],... 

Kinetic studies of NMP kinases, as well as many other enzymes having ATP or other nucleoside triphosphates as a substrate, reveal that these enzymes are essentially inactive in the absence of divalent metal ions such as magnesium (Mg2+) or manganese (Mn2+), but acquire activity on the addition of these ions. In contrast with the enzymes discussed so far, the metal is not a component of the active site. Rather, nucleotides such as ATP bind these ions, and it is the metal ion-nucleotide complex that is the true substrate for the enzymes. The dissociation constant for the ATP-Mg2+ complex is approximately 0.1 mM, and thus, given that intracellular Mg + concentrations are typically in the millimolar range, essentially all nucleoside triphosphates are present as NTP-Mg + complexes. 

Magnesium or Manganese Complexes of Nucleoside Triphosphates Are the True Substrates for Essentially All NTP-Dependent Enzynnes... 

When protein B1 and B2 are mixed in the presence of magnesium ions and dithiothreitol active ribonucleotide reductase with an S2o,w of 9.7S is formed. Stimulatory effectors, such as ATP and TTP, do not effect complex formation. In contrast, in the presence of the negative effector, dATP, at concentrations which inhibit enzyme activity a larger complex is formed with an S20, w of 15.5S. Both complexes contain equimolar amounts of each subunit. A heavy complex is also formed in the presence of mixtures of other nucleoside triphosphates which inhibit enzyme activity. On the other hand the formation of this heavy inactive complex is prevented by ATP at concentrations which reverse the inhibition by dATP (63). More recent experiments (59) have shown that the interaction between proteins B1 and B2 in the presence of dATP is strongly influenced by the presence of sucrose, and indeed in the absence of sucrose subunits B1 and B2 with dATP form a complex with an S20, w of 22.1S. 

Effective controls are omissions of ribosomes, template, nucleoside triphosphates, amino acids and magnesium. Omissions of protein fraction, tRNA and of monovalent ions also inhibit to some extent. Enzyme synthesis depends of course on incubation at biological temperatures. The final yields of enzymes were equal within the temperature range of 28° to 37°, but were progressively less at temperatures below 28°. The speed of synthesis, of course, varies with temperature. Other controls are addition of templates which do not carry the information for the protein to be tested, and the use of various antibiotics. 

This enzyme activity has been observed in myosin and actomyosin, mitochondria, microsomes, and cell membranes. In some cases magnesium ions function as an activator, in others calcium ions, and in still others, both calcium and magnesium are requited. Another form of adenosine-triphosphatase is stimulated by sodium and potassium ions and is inhibited by ouabain. Some forms of the enzyme can hydrolyse inosine triphosphate and other nucleoside-5 -triphosphates. The substrate specificity may depend upon the activating divalent cation and on the presence of monovalent cations. These enzymes are probably important components of a system responsible for facilitating cation transfer in membranes. They should not be confused with adenosine triphosphate pyrophosphatase E.C. 3.6.1.8. 

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