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J. Biol. Chem., Vol. 278, Issue 2, 1174-1185, January 10, 2003
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From the The bifunctional allosteric enzyme HPr
kinase/phosphatase (HPrK/P) from Bacillus subtilis is a key
enzyme in the main mechanism of carbon catabolite repression/activation
(i.e. a means for the bacteria to adapt rapidly
to environmental changes in carbon sources). In this regulation system,
the enzyme can phosphorylate and dephosphorylate two proteins,
HPr/HPr(Ser(P)) and Crh/Crh(Ser(P)), sensing the metabolic state
of the cell. To acquire further insight into the properties of HPrK/P,
electrospray ionization mass spectrometry, dynamic light scattering,
and BIACORE were used to determine the oligomeric state of the protein
under native conditions, revealing that the enzyme exists as a hexamer
at pH 6.8 and as a monomer and dimer at pH 9.5. Using an in
vitro radioactive assay, the influence of divalent cations, pH,
temperature, and different glycolytic intermediates on the activity as
well as kinetic parameters were investigated. The presence of divalent
cations was found to be essential for both opposing activities of the
enzyme. Furthermore, pH values equal to the internal pH of vegetative
cells seem to favor the kinase activity, whereas lower pH values
increased the phosphatase activity. Among the glycolytic intermediates
evaluated, fructose 1,6-diphosphate and fructose 2,6-diphosphate were
found to be allosteric activators in the kinase assay, whereas high concentrations inhibited the phosphatase activity, except for fructose
1,6-diphosphate in the case of HPr(Ser(P)). Phosphatase activity was
induced by inorganic phosphate as well as acetyl phosphate and
glyceraldehyde 3-phosphate. Kinetic parameters indicate a preference
for binding of HPr compared with Crh to the enzyme and supported a
strong positive cooperativity. This work suggests that the oligomeric
state of the enzyme is influenced by several effectors and is
correlated to the kinase or phosphatase activity. The phosphatase
activity is mainly supported by the hexameric form.
Properties and Regulation of the Bifunctional Enzyme HPr
Kinase/Phosphatase in Bacillus subtilis*
§,
,,**
Pharmacologie et Physico-Chimie des
Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Université Louis Pasteur de Strasbourg, Faculté de
Pharmacie, 74 route du Rhin, B.P. 24, F-67401 Illkirch, France and
¶ Laboratoire de Spectrométrie de Masse Bio-Organique, UMR
CNRS 7509, ECPM, Université Louis Pasteur de Strasbourg, 25 rue
Becquerel, F-67087 Strasbourg, France
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by CNRS and Lilly.
**
To whom correspondence should be addressed. Tel.: 33-3-90-24-42-70;
Fax: 33-3-90-24-43-12; E-mail: haiech@pharma.u-strasbg.fr.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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