Magnesium complexes nucleotides

Mn -ATP (from a folded chelate to an extended outer-sphere complex) when the nucleotide binds to pyruvate kinase. It has also been established that the substitution-inert complex Cr iL-ATP binds at the ATP binding site of the pyruvate kinase-M + complex, and studies with this magnetic probe have led to the construction of molecular models for composite complexes of this important enzyme. Steady-state kinetic studies on the Mn +-, Ni +-, and Co +-activated systems suggest that the substrates of pyruvate kinase are PEP, uncomplexed ADP, and free bivalent cations. Magnesium-complexed ADP and ATP bind at the same site on yeast phosphoglycerate kinase, as do the uncomplexed nucleotides. 

A metal-nucleotide complex that exhibits low rates of ligand exchange as a result of substituting higher oxidation state metal ions with ionic radii nearly equal to the naturally bound metal ion. Such compounds can be prepared with chromium(III), cobalt(III), and rhodi-um(III) in place of magnesium or calcium ion. Because these exchange-inert complexes can be resolved into their various optically active isomers, they have proven to be powerful mechanistic probes, particularly for kinases, NTPases, and nucleotidyl transferases. In the case of Cr(III) coordination complexes with the two phosphates of ATP or ADP, the second phosphate becomes chiral, and the screw sense must be specified to describe the three-dimensional configuration of atoms. 

Preliminary rate measurements should allow one to make a plot of initial velocity Vq versus [metal ion], and this should provide information on the optimal metal ion concentration. (For many MgATP -dependent enzymes, the optimum is frequently 1-3 mM uncomplexed magnesium ion.) Then, by utilizing pubhshed values for formation constants (also known as stability constants) defining metal ion-nucleotide complexation, one can readily design experiments to keep free metal ion concentration at a fixed level. To compensate properly for metal ion complexation in ATP-dependent reactions, one must chose a buffer for which a stability constant is known. For example, in 25 mM Tris-HCl (pH 7.5), the stability constant for MgATP is approximately 20,000 M Thus, one can write the following equation ... 

An alkaline pyrophosphatase from rat liver cytoplasm has been partially purified and characterized (24) the corresponding enzyme from mice is inhibited by Mg J+-ADP and free PPj, and free Mg2+ has been implicated as an allosteric activator (23). Partial heat inactivation results in loss of the apparent allosteric effects. Rat liver mitochondrial pyrophosphatase, which is inhibited by adenine nucleotides (36), appears to be bound to the inside of the inner mitochondrial membrane (37). This enzyme, after solubilization, has been separated into two fractions which have somewhat different specificity (24, 38). A pyrophosphatase strongly simulated by sulfhydryl reagents (39) has been partially purified from brain tissue (40). The mono-magnesium PPj complex appears to be the true substrate for this enzyme (41). Pynes and Younathan have purified a pyrophosphatase 1800-fold from human erythrocytes (43). The properties of this enzyme are strikingly similar to those of the yeast enzyme the major difference appears to be the more rigid substrate specificity of the erythrocyte enzyme in the presence of Znz. ... 

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],... 

The function of magnesium in enzyme activity may either be to form a complex with the substrate, as in the magnesium-ATP complex formed in creatine kinase and phosphofhictokinase, or to bind to the enzyme and either produce an allosteric activation or play a direct role in catalysis. If an enzyme is known to utilize a nucleotide as one of its substrates, it can be assumed that magnesium is also required for catalysis. The magnesium ion possibly acts as an electrostatic shield. The enzyme pyravate kinase, described earlier, and shown in Figure 1, requires both magnesium and potassium ions for maximal activity. 

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. 

ALA-dehydratase, and isocitrate dehydrogenase and decreases Na+, K -ATPase activity, Mg +-ATPase activity, and choline uptake into synaptosomes. In vitro, aluminum displaces magnesium from Mg +-ATP complexes, and it could thus antagonize virtually any phosphatetransferring reaction that uses Mg +-nucleotide triphosphate complexes. 

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. 

The coordination structures of manganese nucleotides at enzymic active sites are especially good models for magnesium nucleotides, since they are comparably reactive as substrates. The coordination exchange-inert complexes are somewhat less reactive, but no conflicting results have been obtained to date by these three methods. 

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