title: Structural changes in the catalytic cycle of the Na+,K+-ATPase studied by infrared spectroscopy.
abstract: Pig kidney Na(+),K(+)-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na(3)(+) to an E2P state was initiated by photolysis of P(3)-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg(2+) and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na(+),K(+)-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15 degrees C. Several controls ensured that the observed difference signals were due to structural changes of the Na(+),K(+)-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca(2+)-ATPase and H(+),K(+)-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm(-1), respectively, for alphabeta heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na(3)(+) to an E2P state. A negative band at 1730 cm(-1) is in line with the presence of a protonated Asp or Glu residue that coordinates Na(+) in E1Na(3)(+). Infrared signals were also detected in the absorption regions of ionized carboxyl groups.
authors.affiliation: Max Planck Institute of Biophysics, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt am Main, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Stolz
authors.forename: Erwin
authors.initials: E
authors.lastname: Lewitzki
authors.forename: Rolf
authors.initials: R
authors.lastname: Bergbauer
authors.forename: Werner
authors.initials: W
authors.lastname: Mäntele
authors.forename: Ernst
authors.initials: E
authors.lastname: Grell
authors.forename: Andreas
authors.initials: A
authors.lastname: Barth
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D004372:Ducks; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D010782:Photolysis; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D048251:Squalus acanthias; D013552:Swine
chemical_list: C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase