The formation of a 1-5 phosphodiester linkage in the spontaneous breakdownof 5-phosphoribosyl-alpha-1-pyrophosphate

The decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) in the p resence of Mg2+ at pH=7.8 yields a combination of products including ribose 5-phosphate, ribose 1-phosphate, 5-phosphoribosyl 1,2 cyclic phosphate, in organic phosphate, and pyrophosphate. Hydrogen decoupled P-31 NMR analysis of the product mixture also exhibits a sharp peak (+2.6 ppm from phosphocre atine) in a chemical shift region which includes phosphodiester bonds. Alka line phosphatase treatment of the product mixture results in cleavage of mo nophosphate esters such as ribose I-phosphate and ribose 5-phosphate, but d oes not affect the unidentified peak. Homonuclear (H-1) correlation spectro scopy (COSY) of a partially purified sample was successful in identifying t he hydrogen spectra of this compound. Combined with results from the splitt ing patterns of selectively decoupled P-31 spectra, the COSY data indicate that several hydrogens are directly coupled to the unknown phosphate group with J value matches to the hydrogen on carbon one and to the two hydrogens on carbon Ave. Heteronuclear (H-1-P-31) chemical shift correlation studies confirm these couplings and further substantiate the formation of a ribose 1-5 phosphate linkage during the degradation of PRPP under these condition s. It is presently unknown whether this is an intramolecular or intermolecu lar phosphodiester linkage, although some spectroscopic evidence suggest th e intramolecular bond formation, i.e. a ribose 1,5-cyclic phosphate (R-1,5c P). The formation of R-1,5cP helps explain the observation that the 5-phosp hate group from PRPP becomes labile during the spontaneous degradation of P RPP. (C) 2000 Elsevier Science S.R. All rights reserved.