Stereospecificity of inositol hexakisphosphate dephosphorylation by Paramecium phytase

InsP6 is an abundant compound in many micro-organisms, plants and animal cells. Its function and route of synthesis are still largely unknown. Degradation of InsP6 is mediated by phytase, which in most organisms dephosphorylates InsP6 in a relatively non-specific way. In the micro-organism Paramecium, however, the enzyme has been shown to dephosphorylate InsP6 to InsP2 in a specific order, but its stereospecificity has not been established, i.e. the phosphates are removed in the sequence 6/5/4/3 or 6/5/4/1 or 4/5/6/1 or 4/5/6/3 [Freund, Mayr, Tietz and Schultz (1992) Eur. J. Biochem. 207, 359-367]. We have isolated the InsP4 intermediate and identified its absolute configuration as D-Ins(1,2,3,4)P4. Furthermore, degradation of [3,5-32P]InsP6 yielded a 32P-labelled InsP2 isomer, D-Ins(2,3)P2. These data demonstrate that Paramecium phytase removes the phosphates of InsP6 in the sequence 6/5/4/1. Knowing the stereochemical course of the enzyme, it can be used to elucidate the route of InsP6 synthesis, as it allows us to determine the specific radioactivity at individual positions of the molecular after pulse-labelling cells with [32P]P1 in vivo or [gamma-32P]ATP in vitro.