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

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc 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+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than 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 greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.