Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy.
We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.
Inobe
Tomonao
T
Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Arai
Munehito
M
Nakao
Masaharu
M
Ito
Kazuki
K
Kamagata
Kiyoto
K
Makio
Tadashi
T
Amemiya
Yoshiyuki
Y
Kihara
Hiroshi
H
Kuwajima
Kunihiro
K
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics; D014961:X-Ray Diffraction / Q000379:methods*
D018834:Chaperonin 60; D000255:Adenosine Triphosphate