Structural changes in the catalytic cycle of the Na+,K+-ATPase studied by infrared spectroscopy
2009 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 96, 3433-3442 p.Article in journal (Refereed) Published
Pig kidney Na+,K+-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na3+ to an E2P state was initiated by photolysis of P3-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg2+ 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°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 Ca2+-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 αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na3+ 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 E1Na3+. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.
Place, publisher, year, edition, pages
2009. Vol. 96, 3433-3442 p.
Na+, K+-ATPase, catalytic cycle, infrared spectroscopy
Research subject Biophysics
IdentifiersURN: urn:nbn:se:su:diva-33339DOI: 10.1016%2Fj.bpj.2009.01.010ISI: 000266377100044OAI: oai:DiVA.org:su-33339DiVA: diva2:283022