title: Insights into the functioning of Bacillus subtilis HPr kinase/phosphatase: affinity for its protein substrates and role of cations and phosphate.
abstract: In Bacillus subtilis, carbon catabolite repression is mediated by the HPr kinase/phosphatase (HprK/P) which catalyzes both an ATP-dependent phosphorylation and a dephosphorylation on Ser-46 of either HPr (histidine-containing protein) or Crh (catabolite repression HPr). By using a surface plasmon resonance approach, it was shown here that the presence of magnesium is a prerequisite for the interaction of HprK/P with either HPr or Crh. HprK/P binds both protein substrates with a similar affinity (K(D) of about 40 nM), and addition of nucleotides increases by about 10-fold its affinity for each substrate. In addition, the specificity and the concentration of the cation required for the binding of protein substrates are different from that exhibited by the cation-binding site involved in the nucleotide binding, suggesting the presence of two cation-binding sites on HprK/P. The effects of phosphate on enzymatic activities of HprK/P were also investigated. Phosphate was able to unmask the phosphatase activity, especially in the presence of ATP or both ATP and fructose 1,6-bisphosphate whereas it was shown to inhibit the kinase activity of HprK/P. An apparent competition between phosphate and a fluorescent analogue of nucleotide led to the suggestion that phosphate mediates its effect by binding directly to the ATP-binding site of the enzyme.
authors: Institut de Biologie et Chimie des Protéines, UMR 5086-CNRS, 69367 Lyon Cedex 07, France.
authors: Jean-Pierre
authors: JP
authors: Lavergne
authors: Jean-Michel
authors: JM
authors: Jault
authors: Anne
authors: A
authors: Galinier
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D004789:Enzyme Activation; D004794:Enzyme Repression; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D009097:Multienzyme Complexes; D010710:Phosphates; D010750:Phosphoproteins; D006639:Histidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C110846:YvoE protein, Bacillus substilis; D010749:Phosphoprotein Phosphatases; D008274:Magnesium