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.