Stereochemistry of the hydrolysis of adenosine 5'-thiophosphate catalyzed by venom 5'-nucleotidase

Abstract

The stereochemical problem involving a pro-pro-prochiral phosphorus center, the hydrolysis of 5''-AMP to adenosine and Pi catalyzed by the [Crotalus atrox] venom 5''-nucleotidase, was studied by use of chiral [16O,17O,18O]thiophosphates (Psi). (Rp)- and (Sp)-[.alpha.-18O1]Adenosine 5''-thiophosphates (AMPS) were synthesized by a combined chemical and biochemical procedure. Hydrolysis of (Rp)- and (Sp)-[.alpha.-18O1]AMPS in H217O by 5''-nucleotidase gave 2 enantiomers of chiral Psi of unknown configuration. A 31P NMR method based on the combination of the quadrupolar effect of 17O and the 18O isotope shift was developed to analyze the configuration of chiral Psi. Hydrolysis of (Rp)- and (Sp)-[.alpha.18O1]AMPS in H217O gave (R)- and (S)-[16O,17O,18O]Psi, respectively. The hydrolysis of AMPS catalyzed by the venom 5''-nucleotidase must proceed with inversion of configuration at phosphorus, which suggests that the reaction is most likely an in line single displacement without involving a phosphoryl-enzyme intermediate and without pseudorotation.