Page 90 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
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Enzymes of polyphosphate biosynthesis and degradation
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1990). The purified enzymes from P. shermanii (Phillips et al., 1993) and M. tuberculosis
(Hsieh et al., 1993b) also showed multiple proteins by HPLC gel filtration, native PAGE and
isoelectric focusing (lEF)–PAGE, although a single band was observed by SDS–PAGE. To
explain the existence of multiple forms of glucokinase, it is assumed that the enzyme may
contain residual amounts of strongly bound PolyPs of various chain lengths. As detailed by
Phillips et al., (1999), the enzymes from P. shermanii, M. tuberculosis and Propionibac-
terium arabinosum were all found to be homodimers of 30 kDa sub units.
The common feature of PolyP glucokinases from different sources was that the extracts
containing a polyphosphate glucokinase activity also contained an ATP-dependent activity.
Stable co-purification of these activities suggested that both of them can be catalysed by a
single enzyme (Szymona et al., 1977; Pepin and Wood, 1986). To answer the question of
bifunctionality, Phillips and co-workers carried out extensive purification of the enzymes
from P. shermanii (Phillips et al., 1993), M. tuberculosis (Hsieh et al., 1993a) and P.
arabinosum (Phillips et al., 1999). A detailed characterization of enzyme preparations
unequivocally revealed that a single enzyme from these sources catalyses both polyP- and
ATP-dependent glucokinase activities (Hsieh et al., 1993a; Phillips et al., 1993; Kowalczyk
et al., Phillips et al., 1999).
The most convincing evidence was cloning the gene from M. tuberculosis (Hsieh
et al., 1996a). It was shown that the recombinant protein, expressed and purified from
E. coli, contained both activities. The ability to utilize both inorganic (PolyP) and organic
(ATP) phosphoryl donors in glucose phosphorylation suggested that, due to fundamental
differences in the structures of the two phosphate donors, the residues involved in their
binding may also be different. Horn et al. (1991), Phillips et al. (1993a), and Hsieh et al.
(1993a) provided evidence of separate binding sites for the substrates.
Despitethelackofsequencesimilaritiesbetweeneukaryotichexokinasesandprokaryotic
glucokinases in the putative adenosine site, Hsieh (1996) found some structure similarities
between the adenosine site in polyphosphate glucokinase and the proposed adenosine site in
yeasthexokinase.ComparisonofthekineticfeaturesofPolyP-andATP-dependentreactions
for the enzymes from different sources supports the hypothesis that glucokinase in the earli-
est organisms may have predominantly been dependent on PolyP rather than ATP (Phillips
et al., 1999). There is a progressive decrease in the efficiency of PolyP utilization by glucok-
inases, from older to newer organisms. The polyphosphate glucokinase from Microlunatus
phosphovorus was closely related to the polyphosphate/ATP–glucokinase of Mycobac-
terium tuberculosis, but it could not phosphorylate glucose with ATP (Tanaka et al., 2003).
An enzyme responsible for the PolyP- and ATP-dependent mannokinase activities was
purified to homogeneity from a cell extract of the bacterium Arthrobacter sp. (Mukai et al.,
2003). The enzyme concerned was a monomer with a molecular mass of 30 kDa. This
enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing
PolyP as well as ATP. The catalytic sites for PolyP-dependent phosphorylation and ATP-
dependent phosphorylation of the enzyme were found to be shared, and the PolyP-utilizing
mechanismoftheenzymewasshowntobenon-processive(Mukaietal.,2003).Thededuced
amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of
the PolyP/ATP–glucokinase of M. tuberculosis (level of homology, 45 %), ATP-dependent
glucokinasesofCorynebacteriumglutamicum(45 %),Renibacteriumsalmoninarum(45 %)
and Bacillus subtilis (35 %) (Mukai et al., 2003).
All of these observations suggest a hypothesis that PolyP was a precursor of ATP in
bioenergetic processes at the earliest stage of evolution (Kulaev, 1971, 1974). There might