MAMMALIAN INOSITOL MONOPHOSPHATASE - THE IDENTIFICATION OF RESIDUES IMPORTANT FOR THE BINDING OF MG2-DIRECTED MUTAGENESIS( AND LI+ IONS USING FLUORESCENCE SPECTROSCOPY AND SITE)

The fluorescence properties of residue Trp-219 in inositol monophospha tase are sensitive to the ionization of neighbouring groups. The pH-de pendent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ioni zations in the proximity of Trp-219, namely due to the ionization of H is-217 and Cys-218. By studying the curve of fluorescence intensity ag ainst pH, given by the mutants Cys-218-->Ala or His-217-->Gln, the pK of His-217 was determined to be 7.54 and the pK of Cys-218 was estimat ed to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218-->Al a mutant enzyme is not subject to inhibition by concentrations of Mg2 ions up to 400 mM and has a specific activity of 156 % of the maximum obtainable activity of the native enzyme. The His-217-->Gln mutant en zyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110 % of that obtainable for the native en zyme.