Kinases magnesium ions

ABORTIVE COMPLEXES ADENYLATE KINASE MAGNESIUM ION (INTRACELLULAR)... 

In most animal, plant, and microbial cells, the enzyme that phosphorylates glucose is hexokinase. Magnesium ion (Mg ) is required for this reaction, as for the other kinase enzymes in the glycolytic pathway. The true substrate for the hexokinase reaction is MgATP. The apparent K , for glucose of the animal... 

Beyond the effect of magnesium ion concentration on the equilibrium hydrolysis of adenosine triphosphate to adenosine diphosphate , there is ample evidence that MgATP is generally the most widespread substrate in kinase-type phosphotransferase reactions as well as other ATP-dependent processes. The extent to which MgATP is formed in solution depends on the free (or uncomplexed) magnesium ion concentration, as shown by the following equilibrium constant ... 

Another very important conserved domain in kinases is the activation loop. This 20-30 amino acid region is positioned between a highly conserved DFG and APE motif (IDA region, inter DFG-APE region). In its activated state the activation loop is in an open, extended conformation, which allows substrate binding to the kinase. The aspartate residue (Asp-145) of the DFG motif interacts with one of the two magnesium ions in the active site. 

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. 

Figure 4 Residues within Ga that are critical to the GTP hydrolysis mechanism include arginine-178 and threonine-181 from switch I and glutamine-204 from switch II (colored as in Fig. 2 and numbered as in Ga i coordinates are from PDB record IGFI). Magnesium ion is highlighted in yellow. The planar anion aluminum tetrafluoride, which mimics the y-phosphate leaving group in the hydrolysis reaction, is depicted in metallic red. Note the position of serine-47, the target of phosphorylation by the Y. pestis protein kinase YpkA.

Much structural biology analysis has been performed on protein kinase A (PKA), and its catalytic residues are conserved across the family (6, 7). In PKA, lysine 72 and glutamate 91 orient the y-phosphate toward the protein substrate (Fig. 2b). Aspartate 166 acts as a catalytic base to accept the proton from the hydroxyl nucleophile, and Lys 168 acts as an electrostatic catalyst to stabilize the y-phosphate during the reaction. Asparagine 171 positions a magnesium ion that coordinates the a/fi phosphates (Fig. 2b). 

Figure 2 Snapshots of the overall structure and catalytic machinery of protein kinase A. (a) The overall fold of the catalytic domain is formed by two subdomains, a beta sheet N-terminus (gray) and a C-terminal helical domain (green). ATP binds a cleft between the two lobes, and the phosphoacceptor substrate binds the C-terminal lobe, (b) N-terminal residues Lys 72 and Glu 91 orient the phosphates toward the phosphoacceptor peptide (pink/yellow) in concert with one of two magnesium ions, (c) C-terminal residue Lys 168 acts as an electrostatic catalyst to stabilize the y-phosphate during the reaction while asparagine 171 and aspartate 184 position the phosphates within the active site.

Figure 9.50. ATP-MG2+ Complex Bound to Adenylate Kinase. The magnesium ion is bound to the P and y...

Figure 9.50 ATP-Mg complex bound to adenylate kinase. Nohce that the magnesium ion is bound to the p and 7 phosphoryi groups and to four water molecules at the remaining coordination positions. These water molecules interact with groups on the enzyme, including a conserved aspartate residue. Other interactions have been omitted for clarity.

Figure 2.4. The schematic shows the basis of a tyrosine kinase hTRF assay. The reaction (a) uses a biotinylated peptide substrate that becomes phosphorylated on tyrosine in the presence of kinase, ATP, and magnesium ions. The reaction product is then detected (b).

Kilimann, M.W. Heilmeyer, L.M.G. Multiple activities on phosphorylase kinase. 1. Characterization of three partial activities by their response to calcium ion, magnesium ion, pH, and ammonium chloride and effect of activation by phosphorylation and proteolysis. Biochemistry, 21, 1727-1734 (1982)... 

Nucleoside diphosphate kinases exhibit activity over a wide pH range, but usually have optima at or near pH 7. They require the presence of divalent metals for activity, but have a low specificity in this respect, since Mg +, Mn +, Ca +, Co +, and to a lesser extent, Ni + and Zn +, may satisfy this requirement. Magnesium is evidently the physiological ion serving this enzyme s requirement for a divalent ion. The function of magnesium ion in the nucleoside diphosphate kinase reaction is apparent in the work of Colomb et al. (27), who have shown that for the enzyme from beef heart mitochondria, MgATP, but not free ATP, serves as the phosphate donor. Further, free ADP was shown to be preferred over MgADP as the phosphate acceptor. 

Metal cofactors do not always bind to the enzyme but rather bind to the primary substrate. The resulting substrate-metal complex binds to the enzyme and facilitates its activity. Creatine kinase catalyses the transfer of phosphoryl groups from adenosine triphosphate (ATP), which is broken down to adenosine diphosphate (ADP). The reaction requires the presence of magnesium ions. These, however, do not bind to the enzyme but bind to ATP, forming an ATP Mg complex. It is this complex that binds to the enzyme and allows transfer of the phosphoryl group ... 

As is the case with microsomes and nuclei, amino acid incorporation into isolated mitochondria is dependent upon a proper supply of energy. This requirement can be met partially by added ATP, but much more effectively by supplying substrates for oxidative phosphorylation (such as succinate or a-ketoglutarate plus AMP and phosphate), or by ADP plus a phosphate donor (creatine phosphate or phosphoenolpyruvate) and a kinase (276). Magnesium ions are also needed. 

The reversible reaction required the presence of magnesium ions. Only ATP participated in the reaction it could not be replaced by ADP or adenosine 5 -phosphate. A slight amount of flavin adenine dinucleotide formation was observed when riboflavin instead of riboflavin 5 -phosphate was present, which probably indicates a small contamination with flavo-kinase. 

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