Binding of the activating ion Co(II) to myo-inositol monophosphatase monitored by fluorescence and phosphorescence spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrenemonophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 μM, whereas the apparent activation constant K(u) is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 μM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.