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 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.
Allegrini
S
S
Dipartimento di Scienze del Farmaco, Università di Sassari, via Muroni 23/A, 07100 Sassari Italy.
Scaloni
A
A
Ferrara
L
L
Pesi
R
R
Pinna
P
P
Sgarrella
F
F
Camici
M
M
Eriksson
S
S
Tozzi
M G
MG
D015720:5'-Nucleotidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D007202:Indicators and Reagents; D007555:Isoxazoles; D008024:Ligands; D010455:Peptides; D010710:Phosphates; D011994:Recombinant Proteins; D001224:Aspartic Acid; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D015720:5'-Nucleotidase