On the contribution of stereochemistry to human ITPK1 specificity: Ins(1,4,5,6)P₄ is not a physiologic substrate

Abstract

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

Integration of Inositol Phosphate Signaling Pathways via Human ITPK1

“…In contrast, experiments with hITPK1 indicate a much greater ability to discriminate between substrates, including those that are stereoisomers (16). The inositol phosphate binding site of hITPK1 can be visualized as a strongly electropositive cleft that is stationed at the entrance to the ATP binding pocket (Fig.…”

“…For example, the ligand specificity of hITPK1 depends on the stereochemistry of the inositol ring and the orientation of its hydroxyl groups (16). Most important of all, the eITPK1 structure did not offer any rationalization for how Ins(1,3,4)P 3 can stimulate Ins(1,3,4,5,6)P 5 dephosphorylation by mammalian ITPK1.…”

Molecular basis for the integration of inositol phosphate signaling pathways via human ITPK1

“…The amoeboid ITPK1 is certainly the most promiscuous of the enzymes that metabolize inositol phosphates (Field et al, 2000; Miller et al, 2005), but it would be a truly exceptional enzyme if it lacked all stereochemical specificity. In any case, we have shown that this is absolutely not the case for human ITPK1 (Chamberlain et al, 2007; Riley et al, 2006). We have clearly shown that the determinants of ligand binding include the two-dimensional arrangement of phosphates and hydroxyls around the inositol ring, and also the three-dimensional stereochemistry at each position of the ring.…”

“…In the absence of structural information on enzyme–ligand interactions, we (Ho et al, 2002; Riley et al, 2006) have put forward a proposal which is based on the long-standing observation (Wilcox et al, 1994) that some inositol phosphates may interact with the binding sites of receptors and enzymes in more than one orientation (i.e. “mode”), enabling one inositol phosphate to mimic another by presenting to the docking site some key recognition features.…”

“…We have clearly shown that the determinants of ligand binding include the two-dimensional arrangement of phosphates and hydroxyls around the inositol ring, and also the three-dimensional stereochemistry at each position of the ring. This stereochemically based model is, perhaps, most clearly vindicated by the empirical demonstration that Ins(1,4,5,6) P 4 is not a substrate for ITPK1 (Riley et al, 2006), in contrast to the prediction that arose out of the model of Miller et al (2005).…”