Bovine inositol monophosphatase : Metal ion interactions

Silva, Niroshini Manthri (1999) Bovine inositol monophosphatase : Metal ion interactions. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Inositol monophosphatase is a homodimeric enzyme which is responsible for the recycling of inositol. This is of particular importance in brain tissue since inositol crosses the blood-brain inefficiently hence making dietary inositol effectively unavailable. It catalyses the final step in the de nova synthesis of inositol from glucose. The activity has an absolute requirement for Mg²⁺ ions, although uncompetitive inhibition occurs at high concentrations. The enzyme is also inhibited uncompetitively by Li⁺ ions at therapeutically relevant concentrations of 0.5-1.5 mM, a feature that may underlie the use of Li⁺ in the treatment of manic-depression. Kinetic studies, initially reported a two-Mg²⁺ ion dependency, where a high affinity metal binding site with a K_d of 300μM and a low affinity binding site with a K_m of 3mM need to be saturated for enzyme activity. X-ray crystallographic data have enabled the metal binding residues to be identified and individually mutated (using site directed mutagenesis) in an attempt to assign each residue with either a structural or functional role or both. The two site 2 mutations (D93N and D220N) appeared to cause an increase in affinity for Mg²⁺ at site 1, while the E70Q mutation appears to have abolished Mg²⁺ binding at site 1. The D90N mutant shows a large decrease in affinity for Mg²⁺ at site 2. The activities of all these mutants are extremely low showing that every residue at the active site is important for the catalytic functioning of IMPase. Conformational stability studies of wild type and mutants, D93N, D220N, D90N and E70Q were carried out using natural fluorescent properties of the enzyme. While D93N and D90N showed an increase in stability, D220N and E70Q showed a decrease in stability, with each mutant displaying individual changes in stability in the presence of varying concentrations of Mg²⁺.