Stereospecificity of inositol hexakisphosphate dephosphorylation by Paramecium phytase

InsP(6) is an abundant compound in many micro-organisms, plants and animal cells. Its function and route of synthesis are still largely unknown. Degradation of InsP(6) is mediated by phytase, which in most organisms dephosphorylates InsP(6) in a relatively non-specific way. In the micro-organism Paramecium, however, the enzyme has been shown to dephosphorylate InsP(6) to InsP(2) 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 InsP(4) intermediate and identified its absolute configuration as D-Ins(1,2,3,4)P-4. Furthermore, degradation of [3,5-(32)p]InsP(6) yielded a P-32-labelled InsP(2) isomer, D-Ins(2,3)P-2. These data demonstrate that Paramecium phytase removes the phosphates of InsP(6) in the sequence 6/5/4/1. Knowing the stereochemical course of the enzyme, it can be used to elucidate the route of InsP(6) synthesis, as it allows us to determine the specific radioactivity at individual positions of the molecule after pulse-labelling cells with [P-32]P-i in vivo or [gamma-P-32]ATP in vitro.