Item

Abstract

Changes of protein secondary structure and protein microenvironment associated with partial reactions of Na,K-ATPase have been investigated in detail by reaction-induced FTIR difference spectroscopy. Infrared absorbance changes were recorded in the region from 2000 to 950 cm⁻¹ in H₂O and D₂O. Depending on medium composition, three different partial reactions have been induced by the photochemical release of ATP from different precursors (mainly NPE-caged ATP): (1) ATP binding and release, respectively, at the binding site, (2) formation of the ADP-sensitive phosphoenzyme E1P, and (3) formation of the K⁺-sensitive phosphoenzyme E2P. All partial reactions lead to distinct changes of the infrared spectrum that are characteristic of the adopted states. The observed band amplitudes in the amide I region of the infrared spectrum indicate that the net change of secondary structure change involves at most ∼0.3% of all amino acid residues of Na,K-ATPase. In comparison, the smallest spectral changes are found upon ATP release from the binding site, and the largest for phosphoenzyme conversion (E1P→E2P), indicating larger secondary structure changes. Phosphorylation is accompanied by the appearance of carbonyl bands at 1738 and 1709 cm⁻¹, one of which is tentatively assigned to the phosphorylated Asp³⁶⁹ side chain. Spectral changes in the ranges of 1750–1700 and 1610–1400 cm⁻¹ found upon phosphoenzyme conversion accompanied with Na⁺ release can be attributed to changes in hydrogen bonding, protonation state, and alkali ion coordination of carboxyl groups. The FTIR-difference spectra of Na,K-ATPase and Ca-ATPase (Barth et al. 1996. J. Biol. Chem. 271:30637–30646) differ significantly, probably due to different structural features of these two enzymes.