The fluorescent nucleotides εADP and εATP were used to study the binding and hydrolysis mechanisms of subfragment 1 (S-1) and acto-subfragment 1 from striated and smooth muscle. The quenching of the enhanced fluorescence emission of bound nucleotide by acrylamide analyzed either by the Stern-Volmer method or by fluorescence lifetime measurements showed the presence of two bound nucleotide states for 1-N6-ethenoadenosine triphosphate (εATP), 1-N6-ethenoadenosine diphosphate (εADP), and εADP-vanadate complexes with S-1. The equilibrium constant relating the two bound nucleotide states was close to unity. Transient kinetic studies showed two first-order transitions with rate constants of approximately 500 and 100 s-1 for both εATP and εADP and striated muscle S-1 and 300 and 30 s-1, respectively, for smooth muscle S-1. The hydrolysis of [γ-32P]εATP yielded a transient phase of small amplitude (less than 0.2 mol/site) with a rate constant of 5-10 s-1. Consequently, the hydrolysis of the substrate is a step in the mechanism which is distinct from the two conformational changes induced by the binding of εATP. An essentially symmetric reaction mechanism is proposed in which two structural changes accompany substrate binding and the reversal of these steps occurs in product release. εATP dissociates acto-S-1 as effectively as ATP. For smooth muscle acto-S-1, dissociation proceeds in two steps, each accompanied by enhancement of fluorescence emission. A symmetric reaction scheme is proposed for the acto-S-1 εATPase cycle. The very similar kinetic properties of the reactions of εATP and ATP with S-1 and acto-S-1 suggest that two ATP intermediate states also occur in the ATPase reaction mechanism.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology