Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes

Abstract

The nonspecific phosphohydrolase from E. aerogenes (ATCC 13048) requires divalent metal ions for activity, since Zn present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+ or Ni2+; 6 ions of Zn or Cd can be incorporated into the inactive enzyme and this incorporation of metal ion can be correlated with the regaining of activity. The Cd-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2'',3''-cyclic phosphorothioate (U > pS) to yield uridine 3''-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U > pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz P-31 NMR spectrum of the mixture. The cadmium phosphohydrolase catalyzes hydrolysis of endo-U > pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the P.