The 6‐OH Group of D ‐Inositol 1‐Phosphate Serves as an H‐Bond Donor in the Catalytic Hydrolysis of the Phosphate Ester by Inositol Monophosphatase

Abstract

Inositol monophosphatase plays a pivotal role in the biosynthesis of secondary messengers and is believed to be a target for lithium therapy. It is established how a lithium ion works in inhibiting the enzyme but details of the mechanism for the direct magnesium ion activated hydrolysis of the substrate have been elusive. It is known that substrates require a minimal 1,2-diol phosphate structural motif, which in D-myo-inositol 1-phosphate relates to the fragment comprising the 1-phosphate ester and the 6-hydroxy group. Here it is shown that inhibitors that are D-myo-inositol 1-phosphate substrate analogues possessing 6-substituents larger than the 6-hydroxy group of the substrate, for example, the 6-O-methyl analogue, are able to bind to the enzyme in a congruous manner to the substrate. It is demonstrated, however, that such compounds show no substrate activity whatsoever. It is also shown that a 6-amino group is able to fulfil the role of the 6-hydroxy group of the substrate in conferring substrate activity and that a 6-methylamino group is similarly able to support catalysis. The results indicate that a 6-substituent capable of serving as a hydrogen-bond donor is required in the catalytic mechanism for hydrolysis. It has recently been shown that inositol is displaced from phosphorus with inversion of stereochemistry and we expect that the nucleophilic species is associated with Mg(2+)-1. It is proposed here that the role of the 6-hydroxy group of the substrate is to H-bond with a water molecule or hydroxide ion located on Mg(2+)-2. From this analysis, it appears that the water molecule bound to Mg(2+)-2 serves as a proton donor for the inositolate leaving group in a process that stabilises the alkoxide product and retards the back-reaction.

Removal of the phosphate group in mechanism-based inhibitors of inositol monophosphatase leads to unusual inhibitory activity

“…We have previously demonstrated that 6-alkylamino side chains are tolerated at the active site of IMPase; 6-amino-and 6-methylamino-phosphate derivatives having been shown to be substrates of IMPase. 15,20 Compound 6i is merely the potent inhibitor 3 without a phosphate group attached at C-1. It was hoped that since the phosphate group had been removed then the pendant arm of 3, above all others, would compensate for the binding energy that would be lost through this change.…”

“…[3][4][5][6][7][8] A water molecule associated with Mg 2ϩ 1 deep within the active site acts as a nucleophile and attacks the phosphate group of the substrate -inositol-1-phosphate (-Ins-1-P) 1 and a second water molecule, coordinated to Mg 2ϩ 2 and hydrogen bonded to the 6-OH group of 1 acts as a general acid, protonating the inositolate group as it leaves. 15 Deletion of this 6-OH group removes any substrate activity from related compounds and it is known that the 4-OH and 2-OH groups along with the 1-O atom provide strong interactions for the binding of the substrate to the active site. 16 Many mechanism-based inhibitors of IMPase have been synthesized and evaluated during the accumulation of the above knowledge.…”

ONIOM (DFT:MM) Study of the Catalytic Mechanism of myo-Inositol Monophosphatase: Essential Role of Water in Enzyme Catalysis in the Two-Metal Mechanism

“…Overall, the reaction energy profile (Figure ) suggests that the reaction starts with proton transfer from wat86 to a phosphonyl oxygen atom of Ins(1)P, followed by proton transfer to a carboxylate group of Glu70 and then to the MI oxyanion via a water molecule (wat184). This important role of wat184 in the catalysis was previously proposed by Miller et al and Gill et al The most favorable pathway in Figure involves a barrier of just 26 kcal/mol, which does not seem high enough to exclude the two-metal mechanism. Moreover, we have demonstrated above that no favorable pathway could be described for the three-metal mechanism.…”

“…Wilkie et al also ran MM energy minimization and MD simulation on IMPase and concluded that the reaction follows a noninline displacement mechanism that is accompanied by pseudorotation . A more recent MM study by the same group amended this conclusion and also suggested that a water molecule bound to the Mg 2+ ion at M2 serves as a proton donor . Fujita et al performed docking simulation on human IMPA2 in complex with Mg 2+ ions and Ins(1)P, and proposed that the dephosphorylation reaction proceeds with three Mg 2+ ions .…”

Applications of Nonenzymatic Catalysts to the Alteration of Natural Products

“…Site-selective group transfers to hydroxyl groups are relevant not only for the synthesis of functionalized alcohols but also for their deoxygenation. The removal of specific hydroxyl groups from 13 would allow for biological studies on the importance of the OH functionality at those particular positions. − Toward this end, exchange of the simple acylating agents above for either a phosphoramidite or thiocarbonylating agent − can set up radical-mediated cleavage of its corresponding derivatives (Figure ). Reaction conditions modeled after those of Koreeda were developed for phosphoramidite transfer in the absence of either hydrolysis or oxidation of the resulting P­(III) species .…”