Magnesium complexes nucleosides

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. 

These compounds cause 50% inhibition of a partially purified reductase from Novikoff rat tumor at 10 8 to 10-7 M (150,151). Approximately the same degree of inhibition was observed with other mammalian reductases (152, 153), but the non-heme iron containing reductase from E. coli was not affected. The inhibition of the mammalian reductases is only partially reversible (154). Since these compounds are strong metal chelators complexation of iron is probably involved in the mechanism of inhibition however excess Fe2+ does not reverse the inhibition, and other evidence indicates that these compounds do not act solely by chelating free iron from solution thus depriving the enzyme of a cofactor (150, 151). Kinetic studies indicate no competition with respect to nucleoside diphosphate substrate, nucleotide effector, or magnesium ions, but partial competition for the dithiol substrate was observed. 

In some kinases, such as nucleoside diphosphate kinase, " an intermediate step is the phosphoryl transfer to a group belonging to the enzyme, as happens in ATPase and as was discussed in detail for alkaline phosphatase (Section V.B). In other kinases the phosphoryl transfer occurs directly from the donor to the acceptor in a ternary complex of the enzyme with the two substrates.Often metal ions like magnesium or manganese are needed. These ions interact with the terminal oxygen of the ATP molecule, thus facilitating the nucleophilic attack by the acceptor. The metal ion is often associated with the enzyme. For mechanistic schemes, see the proposed mechanism of action of alkaline phosphatase, especially when a phosphoryl enzyme intermediate is involved. 

---