title: On the mechanism of action of bovine intestinal mucosa 5'-nucleotide phosphodiesterase. Stereochemical evidence for a nucleotidyl-enzyme intermediate.
abstract: The diastereoisomers of adenosine 5'-O-phosphorothioate O-methyl ester have been synthesised. Only the Sp diastereoisomer is a substrate for the 5'-nucleotide phosphodiesterase from bovine intestinal mucosa. The previously unidentified enantiomer of 4-nitrophenyl phenyl phosphonothioate hydrolysed by the enzyme is shown to have the Sp configuration. Digestion of the Sp diastereoisomer of adenosine 5'-O-phosphorothioate O-methyl ester by the enzyme in 18O-labelled water gave 18O-labelled adenosine 5'-O-phosphorothioate which was stereochemically analysed by methylation and subsequent 31P-NMR spectroscopy and shown to possess the Sp configuration. Thus the enzyme-catalysed cleavage proceeded with retention of configuration at phosphorus, presumably via a double-displacement mechanism. This provides strong evidence for the existence of a nucleotidyl-enzyme intermediate on the reaction pathway.
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013237:Stereoisomerism; D013873:Thionucleotides / Q000378:metabolism
chemical_list: D013873:Thionucleotides; C050986:adenosine 5'-O-phosphorothioate O-methyl ester; D000249:Adenosine Monophosphate; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I