Page 203 - The Biochemistry of Inorganic Polyphosphates

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Polyphosphates and polyphosphate-metabolizing enzymes 187
Therefore, a method of glucose determination by immobilized polyphosphate glucoki-
nase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) has been elaborated (Kowal-
czyk and Szymona, 1991). The enzyme was covalently coupled with collagen-coated silica
gel beads and used to determine glucose in serum and other samples, as a packed-bed reac-
tor. The method was based on spectrophotometric measurement of the NADPH produced
by two consecutive reactions, similar to the hexokinase method. The immobilized-enzyme
reactor showed good operational stability during a month of usage, losing about 12 % of its
initial activity (Kowalczyk and Szymona, 1991). An endopolyphosphatase assay using the
same enzyme has also been described (Kowalczyk and Phillips, 1993).
While PolyP is cheaper than AMP, ADP and ATP, it was proposed as a phosphodonor in
enzymatic synthesis. Effective ATP regeneration systems based on polyphosphate kinase
(EC 2.7.4.1, ATP:polyphosphate phosphotransferase) have been elaborated (Butler, 1977;
Murata et al., 1988; Hoffman et al., 1988; Haesler et al., 1992). For example, polyphos-
phate kinase partially purified from E. coli and immobilized on glutaraldehyde-activated
aminoethyl cellulose could carry out the synthesis of ATP from ADP, using long-chain in-
organic PolyP as a phosphoryl donor. Immobilized polyphosphate kinase loses no activity
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when stored in an aqueous suspension for 2 months at 5 C or for 1–2 weeks at 25 C. It
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may be stored indefinitely as a lyophilized powder at −10 C. Storage stability, purity and
yield of its ATP product and low values of the Michaelis constants for its substrates make
it a highly promising enzyme for ATP regeneration (Hoffman et al., 1988).
The overproduction of polyphosphate kinase achieved by recombinant DNA technology
makes the practical use of polyphosphate kinase and PolyP more possible. This activity
of pure polyphosphate kinase enables the practical synthesis of oligosaccharides and their
derivatives (Noguchi and Shiba, 1998; Shiba et al., 2000; Ishige et al., 2001).
For example, galactose 1-phosphate (Gal-1-P) was synthesized with E. coli galactokinase
and an ATP-regeneration system consisting of PolyP and polyphosphate kinase prepared
from ppk-overproducing E. coli cells (Shiba et al., 2000). The phosphorylation efficiency
of galactose with PolyP and polyphosphate kinase was shown to be almost the same as that
with ATP. Via a combined action, polyphosphate kinase and adenylate kinase catalysed the
formation of ADP from AMP, followed by ATP formation from ADP. The addition of adeny-
late kinase to an ATP-regeneration system consisting of PolyP and polyphosphate kinase is
more promising, because cheap AMP can be substituted for expensive ATP or ADP as an
essential compound initially added to the reaction system, and the supplement of adenylate
kinase significantly enhances the efficiency of ATP-regeneration, possibly because ADK
can efficiently catalyse ADP phosphorylation using another ADP as a phospho-donor yield-
ing ATP and AMP. Eventually, with only 4 mM AMP, 28 mM Gal-1-P was synthesized
under the action of polyphosphate kinase and adenylate kinase in the presence of PolyP
(Shiba et al., 2000).
Polyphosphate kinase has been found able to phosphorylate nucleoside diphosphates
to give nucleoside triphosphates, using PolyP as a phosphate donor. Therefore, the pos-
sibility of using PolyP and polyphosphate kinase instead of phosphoenol pyruvate and
pyruvate kinase for enzymatic oligosaccharide synthesis was examined, because PolyP is
quite cheap when compared with phosphoenol pyruvate (Noguchi and Shiba, 1998; Shiba
et al., 2000). Attempts were made to synthesize N-acetyllactosamine (Gal (β1-4) GlcNAc)
using the nucleoside diphosphate kinase-like activity of polyphosphate kinase, where UDP-
Glc pyrophosphorylase and UDP-Glc 4-epimerase catalyse the synthesis of UDP-Glc from