A Fluorescence Investigation of the Nucleotide Binding Sites of the Ca ATPase

Abstract

Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2′[3′]-0-(2,4,6-trinitrophenyl) adenosines'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1, N⁶-ethenoadenosine-5′-diphosphate (ε-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.