Mechanistic studies on myo-inositol monophosphatase
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Enzymic phosphate monoester hydrolysis by inositol monophosphatase from bovine brain (EC 22.214.171.124) occurs 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 Mg2+ ions, one of which is buried and acts as a Lewis acid and phosphate coordination site. The second metal ion appears to coordinate to the alkyl phosphate bridging oxygen, the 'catalytic' hydroxyl group (C-60H of D-Ins 1-P) and to the nucleophile, water or hydroxide. Mechanistic differences have been identified between the hydrolysis of inositol phosphate and nucleoside 2'-monophosphate substrates in that although phosphate-oxygen ligand exchange with the solvent Is facile in the presence of inositol, no such exchange occurs in the presence of adenosine. The minimum structural requirements of a substrate have been demonstrated via synthesis of ethane 1,2-diol monophosphate which shows enzyme activity (Vmax ca. 12% that of Vmax for Ins 1-P, Km = 0.7 mM and Ki = 1.0 mM). Elaboration of the free hydroxyl group to produce (S,R)-, (S)- and (R)-pentane 1,2,5-triol 2-phosphate gave inhibitors of contrasting potency ((S)-pentane 1,2,5-triol 2-phosphate Ki = 0.12 mM, (R)-pentane 1,2,5-triol 2-phosphate, Ki = 3.8 mM) as expected from the structural requirements for hydrolysis to occur. The proposed mechanism of adenosine 2'-monophosphate hydrolysis involving the N3-atom of the adenine moiety has been discounted through spectroscopic analysis of enzymic incubations of new nucleoside substrates (Uridine 2'-monophosphate; Vmax 230% that of Vmax for 2'-AMP, Km = 4.0 mM and 5,6-dihydrouridine 2'-monophosphate; Vmax 70% that of Vmax for 2'-AMP, Km = 1.4 mM), which showed no intermediate phosphates, with only the substrate and final enzymic product (uridine) detected. Ethane 1,2-diol phosphate has been further elaborated to produce 2-methoxyethanol phosphate, diethylene glycol phosphate, pentane 1,5-diolphosphate, diethylene glycol cyclic phosphate and pentane 1,5-diol cyclic phosphate (Ki values range between 3 & 8 mM). Inhibition is attributed to interaction of the substrate with the second Mg2+ ion, and displacement of the catalytic water (hydroxide) molecule. The fact that the cyclic phosphate diesters are not hydrolysed to phosphate monoesters by the enzyme demonstrates that the attacking nucleophile is not positioned on the first (buried) Mg2+ ion. The mechanistic difference of nucleoside 2'-monophosphate hydrolyses is attributed to the ribofuranosyl oxygen acting as a surrogate for the catalytic hydroxyl group of inositol 1-phosphate. Modelling studies have shown that this results in 2'-AMP adopting an unfavourable conformation which is stabilised by the second (catalytic) Mg2+ ion. The absence of the phosphate moiety in adenosine prevents this conformation being achieved at the active site, accounting for the lack of inhibitory activity of adenosine, and the absence of phosphate- oxygen ligand exchange in the presence of adenosine. The proposed mechanism is consistent with all published kinetic data, and the substrate dependency of lithium inhibition.
Thesis, PhD Doctor of Philosophy
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