Purification and Properties of Exopolyphosphatase from the Cytosol of Saccharomyces cerevisiae Not Encoded by the PPX1 Gene

N. A. Andreeva, T. V. Kulakovskaya^*, and I. S. Kulaev

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia; fax: (7-095) 923-3602; E-mail: alla@ibpm.pushchino.ru

^* To whom correspondence should be addressed.

Received May 15, 2003

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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 ~125 kD. A stable enzyme complex with a molecular mass of ~500 kD is composed of two polypeptides of ~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, PP_i, and p-nitrophenyl phosphate. The activity with polyP₃ is 13% of that with polyP₂₀₈. The K_m values for polyP₂₀₈, polyP₁₅, and polyP₃ hydrolysis are 3.5, 75, and 1100 µM, respectively. The enzyme is most active at pH ~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.

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KEY WORDS: polyphosphate, exopolyphosphatase, cytosol, cations, inhibitors, yeast, Saccharomyces cerevisiae

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