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A nucleotidase is a hydrolytic enzyme that catalyzes the hydrolysis of a nucleotide into a nucleoside and a phosphate. For example, they convert adenosine monophosphate to adenosine, and guanosine monophosphate to guanosine.

They have an important function in digestion in that they break down consumed nucleic acids.

They can be divided into two categories, based upon the end which is hydrolyzed:

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5' nucleotidases cleave off the phosphate from the 5' end of the sugar moeity. They can be classified into various kinds depending on their substrate preferences and subcellular localization. Membrane bound 5' nucleotidases displays specificity towards adenosine monophosphates and are predominantly involved in the salvage of preformed nucleotides and in signal transduction cascades involving purinergic receptors. Soluble 5' nucleotidases are all known to belong to the haloacid dehalogenase superfamily of enzymes which are two domian proteins characterised by a modified Rossman fold as the core and variable cap or hood. The soluble forms are further subclassified based on the criterion mentioned above. mdN and cdN are mitochondrial and cytosolic 5'-3' pyrimidine nucleotidases. cN-I is a cytosolic nucleotidase(cN) characterized by its affinity towards AMP as its substrate.cN-II is identified by its affinity towards either IMP or GMP or both. cN-III is a pyrimidine 5' nucleotidase. 5' nucleotidases are involved in varied functions like cell-cell communication, nucleic acid repair, purine salvage pathway for the synthesis of nucleotides,signal transduction, membrane transport etc.

Ref: Hunsucker, S. A., Mitchell, B. S. and Spychala, J. (2005). The 5'-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol. Ther. 107, 1-30.