Probes for the position and mechanistic role of the second ‘catalytic’ magnesium ion in the inositol monophosphatase reaction

Abstract

Two magnesium ions are required for the enzymic hydrolysis of phosphate monoester substrates of inositol monophosphatase. It has been suggested that one (buried) Mg²⁺ ion binds to the enzyme and the phosphate dianion moiety of the substrate through one or more of its negatively charged O-atoms while the second Mg²⁺ ion binds to the substrate bridging phosphate ester O-atom and one other substrate-derived O-atom. This second Mg²⁺ ion may also position and activate the attacking nucleophilic water molecule (A. G. Cole and D. Gani, J. Chem. Soc., Perkin Trans. 1, 1995, previous article). To determine the minimum structural requirements for a substrate, as deduced from the proposed interactions for natural and synthetic substrates with both Mg²⁺ ions, ethane-1,2-diol 1-phosphate was prepared and was found to be a substrate. The design and preparation of a range of minimal structure synthetic probes based on this new substrate including propyl, 2-methoxyethyl, 2-(2-hydroxyethoxy)ethyl and 5-hydroxypentyl monophosphate ester and both antipodes of 1,5-dihydroxypentan-2-yl phosphate allowed the specific interactions between the substrate and the enzyme and/or the second Mg²⁺ ion to be assessed. The results support the proposed roles for the metal ions and provide information on the position of the second Mg²⁺ ion. This information rationalises the properties of known organophosphate substrates and inhibitors for the enzyme and, furthermore, facilitates the construction of a 3-D catalytic mechanism for the inositol monophosphatase reaction which is described. This new catalytic mechanism explains why Li⁺ behaves as an inhibitor and accounts for its unusual inhibitory properties.