Monitoring of secondary and tertiary structure changes in the gastric H+/K+-ATPase by infrared spectroscopy

Conformational changes occurring in the catalytic cycle of the H+/K+-ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged co mpounds were used to release ATP, in the presence of Ca2+, to induce the tr ansition between the E1 and E1-P conformation of the H+/K+-ATPase. In addit ion to bands associated with the photolysis of the caged compounds, some pe aks of the difference infrared spectra were associated with changes in seco ndary structure and modifications of the ionization in the side chains of a mino-acid residues (Glu or Asp). These changes were specific to the reactio n between the ligand and the enzyme. We estimated that 39 amino acids chang ed their secondary structure during the reaction and four amino-acid residu es were deprotonated. Similar spectral changes appeared when ADP was releas ed from its precursor. The release of Pi from the same caged molecule did n ot induce similar changes. Changes in tertiary structure occurring during t he binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total refl ectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were invol ved in a tertiary structure change induced by ATP. This suggested that seco ndary structure change transduced a much larger tertiary structure modifica tion.