Premium
Thermodynamic analysis of F 1 ‐ ATP ase rotary catalysis using high‐speed imaging
Author(s) -
Watanabe Rikiya,
Minagawa Yoshihiro,
Noji Hiroyuki
Publication year - 2014
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2559
Subject(s) - chemistry , catalysis , atp hydrolysis , activation energy , enthalpy , hydrolysis , atp synthase , reaction coordinate , arrhenius equation , reaction rate , reaction mechanism , atpase , crystallography , thermodynamics , enzyme , biochemistry , physics
F 1 ‐ATPase (F 1 ) is a rotary motor protein fueled by ATP hydrolysis. Although the mechanism for coupling rotation and catalysis has been well studied, the molecular details of individual reaction steps remain elusive. In this study, we performed high‐speed imaging of F 1 rotation at various temperatures using the total internal reflection dark‐field (TIRDF) illumination system, which allows resolution of the F 1 catalytic reaction into elementary reaction steps with a high temporal resolution of 72 µs. At a high concentration of ATP, F 1 rotation comprised distinct 80° and 40° substeps. The 80° substep, which exhibited significant temperature dependence, is triggered by the temperature‐sensitive reaction, whereas the 40° substep is triggered by ATP hydrolysis and the release of inorganic phosphate ( P i ). Then, we conducted Arrhenius analysis of the reaction rates to obtain the thermodynamic parameters for individual reaction steps, that is, ATP binding, ATP hydrolysis, P i release, and TS reaction. Although all reaction steps exhibited similar activation free energy values, Δ G ‡ = 53–56 kJ mol −1 , the contributions of the enthalpy (Δ H ‡ ), and entropy (Δ S ‡ ) terms were significantly different; the reaction steps that induce tight subunit packing, for example, ATP binding and TS reaction, showed high positive values of both Δ H ‡ and Δ S ‡ . The results may reflect modulation of the excluded volume as a function of subunit packing tightness at individual reaction steps, leading to a gain or loss in water entropy.