CHARACTERIZATION OF THE SUBSTRATE-INDUCED CONFORMATIONAL CHANGE OF N-IODOACETYL-N'-(5-SULFO-1-NAPHTHYL)ETHYLENEDIAMINE-LABELED SARCOPLASMIC-RETICULUM CA-2+-ATPASE BY USING DIFFERENT KINDS OF SUBSTRATE

Cys-674 of the sarcoplasmic reticulum Ca2+-ATPase was labeled with N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. When Ca2+ was present, the fluorescence intensity and anisotropy decreased greatly upon addition of any substrate used. The observed affinity for each substrate agreed with the previously observed affinity of the catalytic site. The fluorescence drop induced by the adenine nucleotides, ATP and adenosine 5'-(β,γ-methylene)triphosphate (a nonhydrolyzable ATP analog), was much faster than that induced by other substrates. The ATP-induced fluorescence drop preceded phosphoenzyme formation when the ATP concentration was high, but the fluorescence drop coincided with phosphoenzyme formation when it was slowed by reducing ATP concentrations. The fluorescence drop induced by ITP or acetyl phosphate was slow even at high concentrations of the substrate, and it coincided with phosphoenzyme formation. When Ca2+ was absent, the fluorescence intensity and anisotropy decreased only slightly upon addition of any substrate other than the adenine nucleotides. They decreased substantially upon addition of the adenine nucleotides, but the kinetics of this fluorescence drop were quite different from that of the fluorescence drop induced by any substrate in the presence of Ca2+. These results show that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca2+-activated enzyme. This change precedes phosphoenzyme formation in the catalytic cycle and is greatly accelerated by the adenine moiety of the substrate. © 1990.