Kinases ligand exchange

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. 

In most cases the NADH oxidase activates the Na+/H+ exchanger in animal cells. In plant cells, it appears to activate the H+-ATPase. The mechanism for channel activation is analogous to channel activation by ligand receptors. Since the oxidase can control tyrosine kinase, activation of the exchanger is most likely through the MAP tyrosine kinase-activated serine kinase which phosphorylates the antiport. 

In contrast to the other three cations, Mg has a much slower exchange rate of water in its hydration sphere (Table 10.1). Mg often participates in structures, for example, in ATP binding catalytic pockets of kinases and other phosphoryl transferase enzymes, where the metal is bound to four or five ligands from the protein and the ATP. This leaves one or two coordination positions vacant for occupation by water molecules, which can be positioned in a particular geometry by the Mg to participate in the catalytic mechanism of the enzyme. This capacity is an example of outer sphere activation of a substrate by a metal ion (Figure 10.1) as distinct from the... 

A sensor for cAMP requires a conformational change of the protein upon binding of the ligand. This is provided by the two major cAMP-binding proteins the regulatoiy subunit of the cAMP-dependent protein kinase (PKA) or by EPAC (exchange protein directly activated by cAMP). Both proteins were equipped with the standard FRET pair EYFP and ECFP attached to the N- and C-termini, respectively [96]. When expressed in hippocampal neurons or peritoneal macrophages. 

Rates of ligand substitution for Mg(II) and Mn(II) are in the range of lO -lO s". Unless the enzyme has a specific requirement for and is specially designed to prevent dissociation of the metal from its binding site, these ions are expected to rapidly exchange between the bound and free forms in aqueous solutions of the enzyme-metal (EM), substrate-metal (SM), and enzyme-substrate-metal (ESM) complexes. Since Mg(II) and Mn(II) activate a wide variety of enzyme types (e.g., kinases), one can represent all possible equilibria for EMS complex formation as ... 

Co(lII), or Rh(lII), which form inert complexes with nucleotides that exchange ligands on the time scale of days or weeks (especially at low temperatures). It is possible, for example, to separate the A and A isomers of CrATP and use them as substrates in single turnover experiments with various enzymes 23, 24). When the enzyme catalyzes multiple turnovers, the developing circular dichroic (CD) spectrum as one isomer is converted to a product without a CD spectrum can determine the screw-sense specificity. Thus, hexokinase and glycerokinase use the A isomer of CrATP as a substrate, and pyruvate kinase and myokinase (adenylate kinase) use the A isomer (25). The absolute configurations of the ADP and ATP complexes of these metal ions are now known and have been correlated with the CD spectra (26-30). 

---