STEREOSPECIFICITY OF INOSITOL HEXAKISPHOSPHATE DEPHOSPHORYLATION BY PARAMECIUM PHYTASE

InsP(6) is an abundant compound in many micro-organisms, plants and an imal cells. Its function and route of synthesis are still largely unkn own. Degradation of InsP(6) is mediated by phytase, which in most orga nisms dephosphorylates InsP(6) in a relatively non-specific way. In th e micro-organism Paramecium, however, the enzyme has been shown to dep hosphorylate InsP(6) to InsP(2) in a specific order, but its stereospe cificity 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, T ietz and Schultz (1992) Eur. J. Biochem. 207, 359-367]. We have isolat ed 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) y ielded a P-32-labelled InsP(2) isomer, D-Ins(2,3)P-2. These data demon strate that Paramecium phytase removes the phosphates of InsP(6) in th e sequence 6/5/4/1. Knowing the stereochemical course of the enzyme, i t can be used to elucidate the route of InsP(6) synthesis, as it allow s us to determine the specific radioactivity at individual positions o f the molecule after pulse-labelling cells with [P-32]P-i in vivo or [ gamma-P-32]ATP in vitro.