Transient kinetics of substrate binding to [formula omitted] measured by fluorescence quenching

Abstract

This paper examines the transient kinetics of substrate binding to the Na + K + -ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF- Na + K + -ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na + K + -ATPase , in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential ( F( t) = F ∞ + ΔF exp[− Bt α ]). Physically, this function may be interpreted in terms of its inverse Laplace transform Φ( k). which describes a distribution of rate-constants; α reflects the width of this distribution. As TNP-ATP concentration increased, α decreased, reflecting TNP-ATP binding to sites with higher energy barriers. α decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, α was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.