MECHANISM OF THE REACTION CATALYZED BY CYTOSOLIC 5'-NUCLEOTIDASE PHOSPHOTRANSFERASE - FORMATION OF A PHOSPHORYLATED INTERMEDIATE/

Cytosolic 5'-nucleotidase preferentially catalysing the hydrolysis of IMP, GMP and their deoxy derivatives, and endowed with phosphotransfer ase activity, was purified from calf thymus and its reaction mechanism was studied. In the presence of [P-32]IMP, ATP and MgCl2, a covalent enzyme-phosphate intermediate was trapped by mixing with an SDS soluti on. Heat or acid treatment of the enzyme before incubation with radiol abelled substrate prevented formation of the intermediate. Furthermore , on the basis of studies on the kinetic parameters of the enzyme as f unction of pH, and of experiments on thiol oxidation and photo-oxidati on, we suggest the involvement of cysteine and histidine residue(s) in the reaction mechanism.