Studies on 3'-Nucleotidase-nuclease from Potato Tubers

II. Further Studies on Substrate Specificity and Mode of Action

Abstract

1. Hydrolysis of 2'(3')-AMP and 2'(3'), 5'-ADP by potato 3'-nucleotidase-nuclease was studied in detail and the specific attack on nucleoside 3'-phosphates was confirmed.
2. Poly A was degraded principally to 5'-AMP by this enzyme.
3. A time course study of hydrolysis of RNA indicated the endonucleolytic nature of the nuclease.
4. Adenosine 3'-benzylphosphate was hydrolyzed to adenosine and benzylphosphate by this enzyme, while adenosine 5'-benzylphosphate was not a substrate.
5. It is concluded from those findings that the enzyme can recognize nucleoside 3'-phosphate moiety (monoester and diester) as the substrate and cleaves the 3' O-P bond.
6. The kinetic parameters, K_m and V_max, of 3'-nucleotidase-nuclease for 3'-ribonucleotides were determined. Ribonucleosides and 5'-ribonucleotides were competitive inhibitors of this enzyme and their K_i values were determined. RNA and also poly A were potent competitive inhibitors of this enzyme.
7. On the basis of these data, it seemed reasonable to assume that the potato 3'-nucleotidase-nuclease has an active site which includes a nucleoside-binding site and two phosphate-binding sites.