Page 94 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-06
Enzymes of polyphosphate biosynthesis and degradation
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As for eucaryotes, exopolyphosphatases of the yeast Saccharomyces cerevisiae have
been most extensively studied. Exopolyphosphatase activity in a cell homogenate of
S. cerevisiae is high (0.10–0.13 µmol P i per min per mg of protein) (Andreeva et al.,
1994; Wurst and Kornberg, 1994) when compared with bacteria (0.02–0.04 µmol P i per
min per mg of protein) (Akiyama et al., 1993; Bonting et al., 1993b). Exopolyphosphatases
from the cell envelope (Andreeva et al., 1990; Andreeva and Okorokov, 1993), cytosol (An-
dreeva et al., 1996, 1998a, 2003), vacuolar sap (Andreeva et al., 1998b) and mitochondrial
matrix (Lichko et al., 2000) of S. cerevisiae were purified and characterized.
Two polyphosphatases have been purified from an homogenate of S. cerevisiae (Wurst
and Kornberg, 1994; Lorenz et al., 1994b). These enzymes have neutral pH optima, similar
kinetic properties and substrate specificity, and require divalent cations, preferably Mg 2+ or
Co , for the maximal activity. Their activities on tripolyphosphate is nearly 1.5-fold higher
2+
than those on long-chain PolyPs. These enzymes are monomeric proteins, one of 45 kDa
(Wurst and Kornberg, 1994) and the other of 28 kDa (Lorenz et al., 1994b). The properties
of these enzymes are similar to those of cytosolic and cell-envelope exopolyphosphatases
(Andreeva and Okorokov, 1993; Andreeva et al., 1996).
Exopolyphosphatase activities were found and characterized in the purified preparations
of yeast cell nuclei (Lichko et al., 1996, 2002b) and mitochondrial membranes (Lichko
et al., 1998). The presence of specific exopolyphosphatases in these sub-cellular fractions
was confirmed by their insensitivity to inhibitors of other phosphohydrolases occurring in
the cell compartments and differences in pH optima.
All studied exopolyphosphatases of S. cerevisiae exhibited several common features
(Andreeva et al., 1990, 1998 a,b, 2001; Andreeva and Okorokov, 1993; Lichko et al., 1996,
1998, 2000, 2002b, 2003a,b). They hydrolysed PolyPs of various chain lengths with release
of P i , and failed to hydrolyse p-nitrophenylphosphate (the substrate of phosphatases with a
broad spectrum of action), ATP and other nucleoside triphosphates, and PP i. The enzyme–
substrate affinity for all exopolyphosphatases under study was considerably higher with
PolyPs of greater chain lengths (Table 6.3). The sensitivity to a number of inhibitors was
also similar. They were insensitive to molybdate, a commonly used phosphohydrolase in-
hibitor, and fluoride, the inhibitor of pyrophosphatases. They were not inhibited with azide,
oligomycin, orthovanadate, N,N -dicyclohexylcarbodiimide, diethylstilbesterol and nitrate

– the known inhibitors of ATPases of different types. SH reagents such as N-ethylmaleimide
and iodacetamide had little or no effect on exopolyphosphatase activities, showing that the

Table 6.3 The Michaelis constants (µmol) of purified exopolyphosphatases of different
cell compartments of S. cerevisiae (Andreeva and Okorokov, 1993; Andreeva et al., 1998a,b,
2001, 2004; Lichko et al., 1996, 1998, 2000).

Cell compartment

Cytosol
Cell envelope, 40 kDa High-molecular- Vacuolar Mitochondrial
Substrate 40 kDa enzyme enzyme weight enzyme sap matrix
PolyP 15 15 11 75 93 18
0.9 1.2 3.5 2.4 0.25
PolyP 208