Structural difference between two ATP-binding sites of heavy meromyosin revealed by the dynamic fluorescence quenching technique

Acrylamide fluorescence quenching of 1,N⁶-ethenoadenylyl imidodiphosphate (e-AMPPNP) bound reversibly to the ATP binding sites of heavy meromyosin was investigated. In the presence of a 6-fold excess of heavy meromyosin over e-AMPPNP, a modified Stern-Volmer plot was not a linear function of the inverse of the concentration of acrylamide used as a quencher. By analyzing the plot, two Stern-Volmer constants (0.89 M^-1 and 13 M^-1) were obtained for bound e-AMPPNP. About 94% of bound e-AMPPNP had the Stern-Volmer constant of 0.89 M^-1 and 6% of bound e-AMPPNP had the Stern-Volmer constant of 13 M^-1. Moreover, in the presence of a 10-fold excess of e-AMPPNP over heavy meromyosin, a curved Stern-Volmer plot was obtained. It was found from this plot that 50% of bound e-AMPPNP had the Stern-Volmer constant of 13 M^-1 and the remainder had the Stern-Volmer constant of 0.89 M^-1. From these and the above data, it was concluded that one of the two ATP-binding sites of heavy meromyosin binds e-AMPPNP much more tightly than the other site does, and that the fluorescent group of e-AMPPNP bound to the former site is strongly isolated from the solvent as compared with that of e-AMPPNP bound to the latter site.