Investigation of Substrate-binding Sites of Cyclic-ribonucleotide Phosphomutase-5'-phosphodiesterase Using Substrate Analogues

Abstract

The substrate-binding sites of a bifunctional enzyme, cyclic-ribonucleotide phosphomutase-5'-phosphodiesterase from Aspergillus niger, FS-44, were investigated by kinetic studies. A series of adenine nucleotides. inhibited the mutase and diesterase reactions, but adenosine and adenine did not at all. Adenosine monophosphate derivatives acted as competitive inhibitors with respect to both adenosine 3', 5'-cyclic monophosphate in the mutase reaction and adenosine 5'-P-nitrophenyl phosphate in the diesterase reaction with the same decreasing order of inhibition constant. However, the inhibition constants of these compounds in the mutase reaction were 1 order of magnitude lower than those in the diesterase reaction. These results suggest that the active site(s) on the enzyme catalyzing the mutase and diesterase reactions have similar properties, but the substrate-binding sites of both reactions are distinct at least.