Purification and Properties of a Yeast Nucleotide Pyrophosphatase

A nucleotide pyrophosphatase (EC 3.6.1.9) has been purified from extracts of the hybrid yeast, Saccharomyces fragilisxSaccharomyces dobzhanskii, to an extent where it appears homogeneous by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Multiple protein components seen on polyacrylamide and starch gel electrophoresis also exhibit enzyme activity. Ultracentrifugation, Sephadex gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis all gave a molecular weight of approximately 65,000 for the enzyme, the electrophoresis results indicating also that the enzyme consists of a single polypeptide chain. Substrates for the enzyme include several sugar nucleotides (including GDP-mannose and UDP-glucose), pyridine nucleotide coenzymes, and the synthetic substrate, thymidine 5′-p-nitrophenylphosphate. Substrate competition experiments and similar inhibition of hydrolysis of different substrates by metal ion binding agents indicate that a single catalytic site is involved in the hydrolysis of the numerous substrates. No nucleotidase, endonuclease, or exonuclease activities were detectable with the purified enzyme, thus distinguishing it from other yeast and mammalian nucleotide pyrophosphatases which also exhibit nucleotidase and/or exonuclease (phosphodiesterase) activity. The enzyme as isolated does not require addition of any cations for maximal activity, but is strongly inhibited in the presence of metal-binding agents. This inhibition can be reversed by addition of zinc salts or other metal ions, suggesting that protein-bound metal ion is necessary to catalytic activity.