Relationship between the active sites of 2′,3′-cyclic phosphodiesterase with 3′-nucleotidase activity purified from Vibrio alginolyticus

Abstract

Using the purified 2′,3′-cyclic phosphodiesterase (3′-nucleotidase) of slightly halophilic Vibrio alginolyticus, the kinetics of the mutual inhibitions between the substrates and of the inhibitory effects of substrate analogs were studied. The following results were obtained: 1. 1. 3′-Ribonucleotides, 2′-,3′-cyclic ribonucleotides and p-dinitrophenyl phosphate acted as linear competitive inhibitors. 2. 2. The 3′-ribonucleotides all were hydrolyzed at the same active site on the enzyme protein. 3. 3. The activities for 3′ ribonucleotides and for di- p-nitrophenyl phosphate were linear competitively inhibited by ribonucleosides and by 5′-ribonucleotides to a lower degree. The decreasing order of their effectiveness (cytidine, uridine, adenosine and guanosine) was similar to the decreasing order of affinities of the 3′-ribonucleotides (3t'-CMP,3′-UMP, 3′-AMP and 3′-GMP) for the 3′-nucleotidase site. 4. 4. The hydrolysis of di- p-nitrophenyl phosphate was simple linear noncompetitively inhibited by pyrimidine bases; the increasing order of effectiveness was thymine, uracil, and cytosine. In contrast, the hydrolysis of 3′-AMP is linear competitively inhibited by these bases having a reverse order of effectiveness. With the results on the chloride modifications for this enzyme, it was considered that di- p-nitrophenyl phosphate is not hydrolyzed exactly at either the 3′-nucleotidase or the cyclic phosphodiesterase site and that the active sites for di- p-nitrophenyl phosphate and for 3′-nucleotidase possibly overlap at a locus where the ribonucleoside of the 3′-ribonucleotide molecule participates in binding. The patterns for the product inhibition for the active mechanism of the 3−nucleotidase are incompatible with the Ordered Uni Bi mechanism established for the phosphatases, and the existence of a phosphorylated enzyme as an intermediate appears unlikely.