Page 86 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-06
Enzymes of polyphosphate biosynthesis and degradation
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procedure for a glycogen-bound protein of 57 kDa developed by Skorko et al. (1989). No
polyphosphate kinase activity was found in the purified protein, when using recently devel-
oped enzymatic methods of PolyP analysis. Furthermore, no polyphosphate kinase activity
was found associated with any of the proteins bound to the glycogen–protein complex. The
gene corresponding to the 57-kDa protein was cloned and functionally characterized. The
predicted product of the gene did not show similarity to any described ppk but to glyco-
gen synthases instead. In agreement with these results, the protein showed only glycogen
synthase activity (Cardona et al., 2001). It should be noted that PolyP identification in an
earlier paper (Skorko et al., 1989) is based on electrophoresis in polyacrylamide gel, autho-
radigraphy, and subsequent acid hydrolysis or alkali phosphatase hydrolysis of radioactive
spots. Such an assay could not exclude the possibility that the product obtained is not PolyP
but phosphorylated protein(s).
In conclusion, it should be mentioned that PolyP synthesis using ATP or GTP has been
reliably demonstrated in eubacteria only. In many bacteria, polyphosphate kinase is the
main enzyme of PolyP synthesis. The existence of enzymes responsible for PolyP sythesis
using ATP in eucaryotes and archaea is still in question.

6.1.2 3-Phospho-D-Glyceroyl-Phosphate:Polyphosphate
Phosphotransferase (EC 2.7.4.17)
This enzyme, which is also called 1,3-diphosphoglycerate-polyphosphate phosphotrans-
ferase (Kulaev and Bobyk, 1971; Kulaev et al., 1971), catalyses the following reaction:
3-phospho-D-glyceroyl-1-phosphate + PolyP −−→ 3-phosphoglycerate + PolyP
n n + 1
(6.7)

This activity was found first in the Neurospora crassa mutant deficient in adenine, where
the concentrations of ATP and other adenyl nucleotides were sharply reduced (Kulaev and
Bobyk, 1971).
The incubation mixture, which afforded the maximum rate of incorporation of 32 P-
orthophosphate into inorganic PolyP, contained glycilglycine buffer (pH 7.4), MgCl 2 (6
µM), PolyP 75 (0.015 µM), fructose-1,6-diphosphate (5.2 µM), 3-phosphoglyceraldehyde
dehydrogenase (14.4 µg), NAD (8 µM), Na 2 H 32 PO 4 (8 µM) and a cell-free extract of N.
32
crassa. This enzyme system resulted in the incorporation of P into high-molecular-weight
PolyP only. The radioactive product obtained was undialysable and almost completely (80
%) hydrolysed to orthophosphate by treatment with 1 N HCl for 10 min at 100 C. Tri-
◦
cyclophosphate was obtained among the products of incomplete hydrolysis by Thilo and
Wiecker’s method (Thilo and Wiecker, 1957). In order to prove that PolyP synthesis by this
system is a result of glycolytic phosphorylation, the effects thereon of glycolytic and oxida-
tive phosphorylation inhibitors were examined. It was found that iodoacetic acid (12 mM)
and a mixture of sodium arsenate (50 mM) and sodium fluoride (2 mM) inhibited PolyP
biosynthesis in this system by 96 and 95 %, respectively. Inhibitors of oxidative phosphory-
lation, 2,4-DNP (0.014 mM) and sodium azide (0.03 mM) had no effect on the incorporation
32
of P-orthophosphate into the PolyP, but an increase in the concentration to 1 mM retarded
the process by 25%. These results therefore confirm the hypothesis that in such an en-
zyme system PolyP biosynthesis is associated with glycolytic phosphorylation reactions.