PROBING THE ROLE OF METAL-IONS IN THE MECHANISM OF INOSITOL MONOPHOSPHATASE BY SITE-DIRECTED MUTAGENESIS

Since inhibition of myo-inositol monophosphatase (EC 3.1-3.25) by lith ium ions and the resulting attenuation of phosphatidylinositol cycle a ctivity may be the mechanism by which lithium exerts its therapeutic e ffect in the treatment of manic depression, it is of great interest to understand the mechanism of the enzyme and how lithium and other meta ls interact with it. Divalent magnesium is essential for enzyme activi ty, whereas Li+ and high concentrations of Mg2+ act as uncompetitive i nhibitors with respect to substrate. From the recently solved crystal structure of the human enzyme, several amino acid residues in the acti ve site were targeted for mutagenesis studies. Nine single-residue sub stituted mutants were characterized with regard to catalytic parameter s, Mg2+ dependence, and Li+ inhibition. In addition, a terbium fluores cence assay was developed to determine the metal binding properties of the wild-type and mutant enzymes. Although none of these mutations af fected K(m) for substrate substantially, the mutations Glu70-->Gln, Gl u70-->Asp, Asp90-->Asn and Thr95-->Ala, in which residues within coord inating distance of the active site metal were modified, all resulted in large reductions in catalytic activity. The position of Glu70 in th e crystal structure further suggests that this residue may be involved in activating water for nucleophilic attack on the substrate. The mut ations Lys36-->Ile, Asp90-->Asn, Thr95-->Ala, Thr95-->Ser, His217-->Gl n, and Cys218-->Ala all resulted in parallel reductions in both lithiu m and magnesium affinity, suggesting that Li+ and Mg2+ share a common binding site.