A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate 2-Kinase

Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes; from nuclear mRNA export and phosphate homeostasis in yeast and plants, to establishment of left-right asymmetry in zebra fish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the Km values of inositol phosphate substrates of this enzyme, and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar Ki inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases

The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations

The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP6, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P5, describing its interactions in solution with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+, and its solid-formation equilibria with Ca2+ and Mg2+. Ins(1,3,4,5,6)P5 forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe3+ is similar to that of InsP6 and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P5 lacks, is not absolutely necessary for high-affinity Fe3+ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca2+ and Mg2+, Ins(1,3,4,5,6)P5 also forms the polymetallic complexes [M4(H2L)] [where L is fully deprotonated Ins(1,3,4,5,6)P5]. However, unlike InsP6, Ins(1,3,4,5,6)P5 is predicted not to be fully associated with Mg2+ under simulated cytosolic/nuclear conditions. The neutral Mg2+ and Ca2+ complexes have significant windows of solubility, but they precipitate as [Mg4(H2L)]·23H2O or [Ca4(H2L)]·16H2O whenever they exceed 135 and 56 μM in concentration, respectively. Nonetheless, the low stability of the [M4(H2L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P5 even under significant Mg2+ or Ca2+ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P5 in straightforward plots

Simple synthesis of 32P-labelled inositol hexakisphosphates for study of phosphate transformations

In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with 32P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates

Regioselective Opening of <i>myo</i>-Inositol Orthoesters: Mechanism and Synthetic Utility

Acid hydrolysis of <i>myo</i>-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-<i>O</i>-acyl <i>myo</i>-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-<i>O</i>-acyl inositol 1,3,4,5,6-pentakisphosphates and <i>myo</i>-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-<i>O</i>-C­(O)­CD₂R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester <i>myo</i>-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-<i>O</i>-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments