MECHANISM OF INOSITOL MONOPHOSPHATASE, THE PUTATIVE TARGET OF LITHIUM-THERAPY

myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolas e, EC 3.1.3.25) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depressi on therapy. The x-ray crystallographic structure of human inositol mon ophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, thre e ions were present with one being displaced upon phosphate binding. W e report here modeling, kinetic, and mutagenesis studies on the enzyme , which reveal the requirement for two metal ions in the catalytic mec hanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling s tudies based on the various x-ray crystallographic structures (includi ng those with Gd3+ and substrate bound) further support a two-metal me chanism and define the positions of the two metal ions relative to sub strate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, a ppears to act as a Lewis acid, stabilizing the leaving inositol oxyani on. In this model, the 6-OH group of substrate acts as a ligand for th is second metal ion, consistent with the reduced catalytic activity ob served with substrate analogues lacking the 6-OH. Evidence from Tb3+ f luorescence quenching and the two-metal kinetic titration curves sugge sts that Li+ binds at the site of this second metal ion.