title: The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding.
abstract: Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.
authors.affiliation: Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, 69367 Lyon Cedex 07, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Jault
authors.forename: S
authors.initials: S
authors.lastname: Fieulaine
authors.forename: S
authors.initials: S
authors.lastname: Nessler
authors.forename: P
authors.initials: P
authors.lastname: Gonzalo
authors.forename: A
authors.initials: A
authors.lastname: Di Pietro
authors.forename: J
authors.initials: J
authors.lastname: Deutscher
authors.forename: A
authors.initials: A
authors.lastname: Galinier
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology*; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D005635:Fructosediphosphates; D007454:Iodides; D009711:Nucleotides; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; C029063:fructose-1,6-diphosphate