Expression and Characterization of Recombinant Bovine Cytosolic 5′-Nucleotidase IMP-GMP Specific

Abstract

Cytosolic 5′-nucleotidase specific for IMP-GMP and their respective deoxyderivatives (5′N) is an ubiquitous enzyme able to catalyse the hydrolysis of purine nucleoside monophosphates or the transfer of the phosphate to an acceptor nucleoside. The enzyme has been purified from several sources and its kinetic parameters and molecular characteristics have been studied.^1,2 All the cytosolic 5′-nucleotidases described, although sometimes differ remarkably in molecular mass, exhibit very similar properties, such as the activation by ATP and the inhibition by orthophosphate (Pi). A reaction mechanism proceeding via the formation of a covalent enzyme-phosphate intermediate was proposed by Worku and Newby.³ The validity of this mechanism was recently demonstrated by the isolation of an enzyme-phosphate covalent complex. Even though there is indirect evidence for an involvement of histidine and cysteine residues in catalysis, nothing is known about the nature of the amino acid acceptor of the phosphate.⁴