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.

Figure 1

Comparison of uncorrected Na⁺,K⁺-ATPase spectra (solid lines) and normalized photolysis spectra (dashed lines) in the presence of 130 mM NaCl and 3 mM free Mg²⁺, recorded 0.66–3.24 s after the photolysis flash at 15°C. (a) Unlabeled caged ATP. (b) [¹⁵N] Caged ATP. The spectra are an average of three experiments performed with different samples.

Figure 2

Photolysis-corrected spectra of Na⁺,K⁺-ATPase in the presence of 130 mM NaCl and 3 mM free Mg²⁺, recorded at 15°C. (a) The full-line and dashed-line spectra were recorded 0.66–3.24 s and 400 s after the photolysis flash, respectively. (b) Control spectrum with FITC-labeled ATP recorded 0.66–3.24 s after the photolysis flash.

Figure 3

Photolysis-corrected spectra of Na⁺,K⁺-ATPase in the presence of 130 mM NaCl, 3 mM free Mg²⁺, and 20 mM KCl recorded at 5°C, (a) 0.66–3.24 s and (b) 320 s after the photolysis flash. (c) Control sample containing 4 mM vanadate. Spectrum recorded 16 s after the photolysis flash. The spectra are an average over three experiments performed with different samples.

Figure 4

Measurement of hydrolytic activity in infrared samples at 5°C. The integrated 1246 cm⁻¹ band is plotted against time. Selected spectra of this experiment are shown in Fig. 3. Full circles: Na⁺,K⁺-ATPase in the presence of 130 mM NaCl, 3 mM free Mg²⁺, and 20 mM KCl. Open circles: Control sample containing 4 mM vanadate.