Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis.
The fluorescence properties of residue Trp-219 in inositol monophosphatase are sensitive to the ionization of neighbouring groups. The pH-dependent changes in the fluorescence emission intensity and wavelength of maximum emission appear to arise as the result of two separate ionizations in the proximity of Trp-219, namely due to the ionization of His-217 and Cys-218. By studying the curve of fluorescence intensity against 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 estimated to be about 8.2. These mutants have altered kinetic parameters for catalytic Mg2+ ions and inhibitory Mg2+ and Li+ ions. The Cys-218-->Ala 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 enzyme shows reduced sensitivity to inhibition by Mg2+ and Li+ ions, and has a specific activity of 110% of that obtainable for the native enzyme.
Gore
M G
MG
Department of Biochemistry, School of Biological Sciences, University of Southampton, U.K.
Greasley
P
P
McAllister
G
G
Ragan
C I
CI
D001483:Base Sequence; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009838:Oligodeoxyribonucleotides; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D008274:Magnesium