Does Fluorescence of 4-Nitrobenzo-2-Oxa-l, 3-Diazole Incorporated into Sarcoplasmic Reticulum ATPase Monitor Putative E1-E2 Conformational Transition?1

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Abstract
When a purified preparation of sarcoplasmic reticulum Ca2+-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-l, 3-diazole (NBD-C1) in 1 mM AMPPNP and 1 mM CaCl2 at 25°C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C122E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2++ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2+-ATPase reaction.