Bovine cytosolic 5 '-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate

Cytosolic; 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'- mononucleotide hydrolysis or be transferred to an appropriate nucleoside ac ceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiol ogical inhibitors. Peptide mapping experiments with the phosphoenzyme previ ously treated with tritiated borohydride allowed isolation of a radiolabele d peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52 -phosphate intermediate. Site-directed mutagenesis experiments confirmed th e essential role of Asp-52 in the catalytic machinery of the enzyme and sug gested also that Asp-54 assists in the formation of the acyl phosphate spec ies. From sequence alignments we conclude that cytosolic 5'-nucleotidase, a long with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.