Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca2+-ATPase catalytic site

• Published in: Biochimica et biophysica acta, Protein structure and molecular enzymology Vol. 1118; no. 3; pp. 231 - 238
• Main Authors: Ivkova, M.N., Pletnev, V.V., Vinokurov, M.G., Pechatnikov, V.A., Ivkov, V.G., Jona, I., Fölöp, J., Köver, A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.02.1992; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; ATPase, Ca2; Biological and medical sciences; Catalytic site; Enzymes and enzyme inhibitors; Fluorescein isothiocyanate; Fundamental and applied biological sciences. Psychology; Hydrolases; Lanthanide binding; Sarcoplasmic reticulum; Fluorescein isothiocyanate; Mops; FITC; Sarcoplasmic reticulum; FIT-SR; Catalytic site; ATPase, Ca2; Lanthanide binding; EGTA; SR; Tris; Ca-ATPase; Vertebrata; Mammalia; Calcium; Enzyme; Active site; Rabbit; pH; Lagomorpha; Striated muscle; Conformation
• ISSN: 0167-4838; 1879-2588
• DOI: 10.1016/0167-4838(92)90280-Q

The conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum (SR) Ca2+-ATPase have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC) bound to the adenine subsite. The FITC-SR fluorescence parameters have been examined in the pH range 5.7–8.0 in the presence of EGTA, Ca2+ or Ln3+ (La3+, Pr3+, Nd3+, Tb3+ etc.). A quantitative method to calculate the equilibrium between the protein conformers is proposed on the basis of the fluorometric titration curve analysis. The distance Nd3+-FITC was estimated to be about 1 nm at pH 6–7 and 1.7 nm at pH 8 which can be interpreted as an increase of the distance between the nucleotide and phosphorylation domains of Ca2+-ATPase in alkaline media. These studies suggest that the ligand-stabilized E1-form of Ca2+-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7–8.0. The pH-dependence of the ratio of these states correlates with that of the E1 ↔ E2 equilibrium without ligands. These dependences were approximated by simple Henderson-Hassellbach equations with pK 7.0 ± 0.1, i.e. the transition between two protein conformations is probably governed by one proton dissociation.