title: Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity.
abstract: The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and Km(ATP) values of 0.3 min(-1) and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.
authors.forename: Veronika
authors.initials: V
authors.lastname: Fojtikova
authors.forename: Martin
authors.initials: M
authors.lastname: Stranava
authors.affiliation: §Laboratoire d'Optique et Biosciences, INSERM U1182-CNRS UMR7645, Ecole Polytechnique, 91128 Palaiseau Cedex, France.
authors.forename: Marten H
authors.initials: MH
authors.lastname: Vos
authors.affiliation: §Laboratoire d'Optique et Biosciences, INSERM U1182-CNRS UMR7645, Ecole Polytechnique, 91128 Palaiseau Cedex, France.
authors.forename: Ursula
authors.initials: U
authors.lastname: Liebl
authors.forename: Jakub
authors.initials: J
authors.lastname: Hranicek
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kitanishi
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
authors.forename: Marketa
authors.initials: M
authors.lastname: Martinkova
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D004789:Enzyme Activation; D005914:Globins / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D006418:Heme; D002248:Carbon Monoxide; D000255:Adenosine Triphosphate; D005914:Globins; D007501:Iron; D011494:Protein Kinases; D000071677:Histidine Kinase; D010100:Oxygen