Abstract
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 acceptor, 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 physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled 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 the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.
Highlights
Hydrolysis of the phosphate esterified in 3Ј or 5Ј of mononucleotides is catalyzed by a family of nucleotidases whose members differ in terms of substrate specificity, cellular location, regulation, distribution, and amino acid sequence
5Ј-Nucleotidase Inactivation by Woodward’s Reagent K—To get information on the nature of the amino acid residues occurring in the active site of 5Ј-nucleotidase, different inhibitors were tested as effectors of the enzyme activity
The partial protection exerted by substrates and inhibitors of the enzyme is strongly indicative that some of the modified residues are located in the active site
Summary
Hydrolysis of the phosphate esterified in 3Ј or 5Ј of mononucleotides is catalyzed by a family of nucleotidases whose members differ in terms of substrate specificity, cellular location, regulation, distribution, and amino acid sequence. A DXDX(T/V) motif has been described as being involved in the catalytic mechanism of a number of other phosphatases/phosphotransferases (15), suggesting a common mechanism of action for all these enzymes that do not present any significant sequence homology with cN-II. Sequence Comparison—The result mentioned above demonstrated that Asp-52 is the amino acid residue of cN-II that is phosphorylated during catalysis; in addition, Asp-54 seems to assist in the formation of this covalent intermediate.
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