Mechanistic and structural studies on myo-inositol monophosphatase: the emerging target for lithium therapy
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Enzymic phosphate monoester hydrolysis by myo-inositol monophosphatase from bovine brain (EC.22.214.171.124) 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.
Thesis, PhD Doctor of Philosophy
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