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 compounds were used to release ATP, in the presence of Ca2+, to induce the transition between the E1 and E1-P conformation of the H+/K+-ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of Pi from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.
Scheirlinckx
F
F
Service de Structure et Fonction des Membranes Biologiques, Université Libre de Bruxelles, Belgium.
Buchet
R
R
Ruysschaert
J M
JM
Goormaghtigh
E
E
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D010782:Photolysis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013552:Swine
D008024:Ligands; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase