PROBES FOR THE POSITION AND MECHANISTIC ROLE OF THE 2ND CATALYTIC MAGNESIUM-ION IN THE INOSITOL MONOPHOSPHATASE REACTION

Two magnesium ions are required for the enzymic hydrolysis of phosphat e monoester substrates of inositol monophosphatase. It has been sugges ted that one (buried) Mg2+ ion binds to the enzyme and the phosphate d ianion moiety of the substrate through one or more of its negatively c harged O-atoms while the second Mg2+ ion binds to the substrate bridgi ng phosphate ester O-atom and one other substrate-derived O-atom. This second Mg2+ ion may also position and activate the attacking nucleoph ilic water molecule.(A. G. Cole and D. Gani, J. Chem. Sec., Perkin Tra ns. 1, 1995, previous article) To determine the minimum structural req uirements for a substrate, as deduced from the proposed interactions f or natural and synthetic substrates with both Mg2+ ions, ethane-1,2-di ol 1-phosphate was prepared and was found to be a substrate. The desig n and preparation of a range of minimal structure synthetic probes bas ed on this new substrate including propyl, 2-methoxyethyl, 2-(2-hydrox yethoxy)ethyl and 5-hydroxypentyl monophosphate ester and both antipod es of 1,5-dihydroxypentan-2-yl phosphate allowed the specific interact ions between the substrate and the enzyme and/or the second Mg2+ ion t o be assessed. The results support the proposed roles for the metal io ns and provide information on the position of the second Mg2+ ion. Thi s information rationalises the properties of known organophosphate sub strates and inhibitors for the enzyme and, furthermore, facilitates th e construction of 3-D catalytic mechanism for the inositol monophospha tase reaction which is described. This new catalytic mechanism explain s why Li+ behaves as an inhibitor and accounts for its unusual inhibit ory properties.