Purification and properties of exopolyphosphatase from the cytosol of Saccharomyces cerevisiae not encoded by the PPX1 gene

• Published in: Biochemistry (Moscow) Vol. 69; no. 4; pp. 387 - 393
• Main Authors: Andreeva, N A, Kulakovskaya, T V, Kulaev, I S
• Format: Journal Article
• Language: English
• Published: United States Springer 01-04-2004; Springer Nature B.V
• Subjects: Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - isolation & purification; Acid Anhydride Hydrolases - metabolism; Brewer's yeast; Cations; Cytoplasm; Cytosol; Cytosol - enzymology; Enzyme Inhibitors - chemistry; Enzyme Stability; Enzymes; Hydrogen-Ion Concentration; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Yeast; Yeasts
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1023/B:BIRY.0000026193.44046.29

A novel exopolyphosphatase has been isolated from the cytosol of Saccharomyces cerevisiae grown to the stationary phase after its transfer from phosphate-deficient to complete medium. The PPX1 gene responsible for 40-kD exopolyphosphatase of the cytosol does not encode it. Specific activity of the preparation is 150 U/mg, purification degree is 319, and the yield is 16.9%. The minimal molecular mass of the active but unstable enzyme complex is approximately 125 kD. A stable enzyme complex with a molecular mass of approximately 500 kD is composed of two polypeptides of approximately 32 and 35 kD and apparently polyphosphates (polyP). Unlike the enzyme encoded by PPX1, the high-molecular-mass exopolyphosphatase is slightly active with polyP3, not inhibited by antibodies suppressing the activity of 40-kD exopolyphosphatase, inhibited by EDTA, and stimulated by divalent cations to a lesser extent. The high-molecular-mass exopolyphosphatase hydrolyzes polyP with an average chain length of 208 to 15 phosphate residues to the same extent, but is inactive with ATP, PPi, and p-nitrophenyl phosphate. The activity with polyP3 is 13% of that with polyP208. The Km values for polyP208, polyP15, and polyP3 hydrolysis are 3.5, 75, and 1100 microM, respectively. The enzyme is most active at pH approximately 7. Co2+ at the optimal concentration of 0.1 mM stimulates the activity 6-fold, while Mg2+ at the optimal concentration of 1 mM enhances it 2-fold. The enzyme under study is similar in some properties to an exopolyphosphatase purified earlier from yeast vacuoles.