Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

Changes in the fluoresence ofN-acetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca²⁺-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 andEP 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-sensitiveEP, and transition of ADP-sensitiveEP to ADP-insensitiveEP. 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 Ca²⁺-binding sites in the transmembrane domain in these latter two steps.Abbreviations SR sarcoplasmic reticulum - EP phosphoenzyme - EDANS N-acetyl-N-(5-sulfo-1-naphthyl)ethylenediamine - AMP-PCP adenosine 5-(, -methylene)triphosphate - NEM N-ethylmaleimide