Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca(2+)-ATPase.
Changes in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca(2+)-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium.enzyme.substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca(2+)-binding sites in the transmembrane domain in these latter two steps.
Kanazawa
T
T
Department of Biochemistry, Asahikawa Medical College, Japan.
Suzuki
H
H
Daiho
T
T
Yamasaki
K
K
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000252:Calcium-Transporting ATPases