Mechanistic and structural studies on myo-inositol monophosphatase: the emerging target for lithium therapy

Abstract

Enzymic phosphate monoester hydrolysis by myo-inositol monophosphatase from bovine brain (EC.3.1.3.25) has been shown to occur via the direct displacement of phosphate by water rather than by a two step mechanism involving a phosphorylated enzyme intermediate. The catalytic process is believed to involve two magnesium ions, one of which is buried deep in the active-site cleft (Mg1) and co-ordinated the phosphate moiety of the substrate. The second metal ion (Mg2) is located closer to the opening of the active site and co-ordinates to the alkyl-phosphate bridging oxygen bond of the substrate. Detailed chemical and kinetic studies on the enzyme have defined many of the interactions of the natural substrate for the enzyme, inositol 1-phosphate, and has led to the proposal that Mg2, which is readily accessible from bulk solvent, should position and activate the nucleophilic water molecule through chelation. Independent X-ray crystallographic studies of protein-substrate complexes using inhibitory metal ions to prevent reaction has provided an almost identical picture of the active-site interactions. However, this study has placed the nucleophilic water molecule on Mg1. The two different sites for the nucleophile would give different stereochemical courses for phosphoryl transfer. A location on Mg1 would give inversion of configuration through an in-line displacement whereas attack by a water molecule chelated to Mg2 would give retention via a novel adjacent association and pseudorotation. In order to distinguish between these two mechanisms, the stereochemical course of the inositol monophosphatase reaction must be determined with respect to the phosphorus centre. A synthetic route has been developed to produce the chirally labelled substrate analogues of inositol 1-phosphate which are required to perform such a study. These compounds, (RP)- and (5P)-inositol l-[18O]phosphorothioate can now be produced with a high degree of isotopic enrichment. The absolute configuration at the phosphorus centre of each individual enantiomer has been determined indirectly using single crystal X-ray analysis. Preliminary work has also been carried out on the hydrolysis of these substrate analogues by inositol monophosphatase using [17O] water and the subsequent derivatisation of the chiral inorganic [16O, 17O, 18O] phosphorothioate product. Derivatisation of the inorganic phosphorothioate product is necessary in order to determine its absolute configuration at phosphorus. A comparison of this result with the known configuration of the substrate analogue which was processed, will allow the stereochemical course of the inositol monophosphatase reaction to be determined.