Stereochemistry of hydrolysis by snake venom phosphodiesterase.

18OAdenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled 18Oadenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to 18Oadenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging 1,2-mu-18O or nonbridging 1-18O was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.