Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

J. Biol. Chem. 278 (2): 1174-1185

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Properties and Regulation of the Bifunctional Enzyme HPr Kinase/Phosphatase in Bacillus subtilis *

Helena Ramström{ddagger}§, Sarah Sanglier||, Emmanuelle Leize-Wagner, Claude Philippe{ddagger}, Alain Van Dorsselaer, , and Jacques Haiech{ddagger}**

From the {ddagger}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

ABSTRACT Back to Top

Abstract: 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.


Received for publication September 4, 2002.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Carbon Catabolite Repression in Bacillus subtilis: Quantitative Analysis of Repression Exerted by Different Carbon Sources.
K. D. Singh, M. H. Schmalisch, J. Stulke, and B. Gorke (2008)
J. Bacteriol. 190, 7275-7284
   Abstract »    Full Text »    PDF »
Phenolic Acid-Mediated Regulation of the padC Gene, Encoding the Phenolic Acid Decarboxylase of Bacillus subtilis.
N. P. Tran, J. Gury, V. Dartois, T. K. C. Nguyen, H. Seraut, L. Barthelmebs, P. Gervais, and J.-F. Cavin (2008)
J. Bacteriol. 190, 3213-3224
   Abstract »    Full Text »    PDF »
The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators.
A. H. Klein, A. Shulla, S. A. Reimann, D. H. Keating, and A. J. Wolfe (2007)
J. Bacteriol. 189, 5574-5581
   Abstract »    Full Text »    PDF »
How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria.
J. Deutscher, C. Francke, and P. W. Postma (2006)
Microbiol. Mol. Biol. Rev. 70, 939-1031
   Abstract »    Full Text »    PDF »
Unfolding of Rabbit Muscle Creatine Kinase Induced by Acid: A STUDY USING ELECTROSPRAY IONIZATION MASS SPECTROMETRY, ISOTHERMAL TITRATION CALORIMETRY, AND FLUORESCENCE SPECTROSCOPY.
Y. Liang, F. Du, S. Sanglier, B.-R. Zhou, Y. Xia, A. Van Dorsselaer, C. Maechling, M.-C. Kilhoffer, and J. Haiech (2003)
J. Biol. Chem. 278, 30098-30105
   Abstract »    Full Text »    PDF »
HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate.
S. Nessler, S. Fieulaine, S. Poncet, A. Galinier, J. Deutscher, and J. Janin (2003)
J. Bacteriol. 185, 4003-4010
   Full Text »    PDF »

To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882