CONFORMATIONAL CHANGES OF THE SARCOPLASMIC RETICULUM Ca–ATPase INDUCED BY SUBSTRATE BINDING AND PHOSPHORYLATION

Abstract

Publisher Summary The present image of the Ca-ATPase functioning is essentially derived from studies using more standard enzymological approaches. The concept of enzyme conformational changes plays an important role in the models advocated by researchers in this field; first, the simplest intuitive scheme of transport involves an alternate sites mechanism where the transport sites are alternatively exposed to both sides of the membrane with a simultaneous change in binding constant for the ion transported and second, in most of the schemes published, the enzyme conformation provides the link between the ions translocation reaction and the phosphate transfer reactions. These two key roles of the enzyme conformation change are the basis for the so-called E ⇆ E* scheme of the Ca-ATPase and the closely related E1 ⇆ E2 scheme of the (Na-K)-ATPase. Attempts to detect these putative conformational changes of the Ca-ATPase and (Na-K)-ATPase were numerous: the measurements of the reactivity of active sites, chemical modifications, susceptibility of SH groups or to tryptic attack, the use of optical properties of extrinsic probes, or the measurement of the protein intrinsic fluorescence changes. This chapter proposes and discusses a general interpretation of these fluorescence changes. Combined with accurate static and time resolved titration of active sites with labeled substrates, the recording of the intrinsic fluorescence changes provides one of the best method presently available to study the events in the life of the Ca-ATPase enzyme.