Inositol monophosphatase is a homodimeric enzyme that catalyses the dephosphorylation of inositol monophosphates to inositol and inorganic phosphate. It has a dual role in that it recycles the inositol, which is critical in brain tissue since it lacks an adequate import system for this moiety and secondly it carries out the final step in the de novo conversion of glucose to inositol. The enzyme has an absolute requirement for Mg^^ ions for catalytic activity although uncompetitive inhibition takes place at high concentrations. In addition, the enzyme is inhibited uncompetitively by Li^ ions. Kinetic studies have reported the existence of two metal binding sites, a high affinity site with a Kd of 300pM and a lower affinity site with a Km value of 3mM. The X-ray structure of the human enzyme has provided much insight into the structural features required for Mg^^ and substrate binding. Based on these findings, corresponding residues in the structurally homologous bovine enzyme located within loop regions, near the active site and at the dimer interface were mutated. Several spectroscopic techniques were employed to determine if these residues were structurally or functionally significant. A G76S mutant displayed unchanged metal binding affinities for both sites 1 and 2. The mutant G69S displayed reduced metal binding affinity at both sites and also for substrate. The stability of this mutant with respect to the denatured state was significantly lower than that of the WT enzyme. The mutation H188Q at the dimer interface displayed a similar site 1 IQ to the WT enzyme, however, its specific activity was markedly increased; it displayed decreased affinity for both Mg^^ binding to site 2 and for substrate. The E30P mutant displayed an increased affinity for Mg^^ binding at sitel and for substrate, but the affinity for Mg^"^ binding at site 2 was unchanged. Mutant C218D displayed a 3-fold and 2-fold decrease in affinity at sites 1 and 2 respectively. Crystals of WT BIMP were grown for the first time, in the presence of the natural Mg^^ ligand. The X-ray structure has been successfully determined at 1.6A in the laboratory of Professor Steve Wood in Southampton. Three Mg'^ binding sites are present in each subunit of the BIMP dimer. The Mg^^ binding sites in subunit B are identical to those seen in the Mn^^ and Ca^^ bound human enzyme structures. However, the third site for Mg^^ in subunit A is differently located and only partially occupied. The significance of the third Mg^^ binding site of BIMP in the proposed mechanism of the enzyme is discussed.