Transient kinetic analysis of ATP-induced allosteric transitions in the eukaryotic chaperonin containing TCP-1.
The chaperonin CCT (chaperonin containing t-complex polypeptide 1 (TCP-1)) from bovine testis was mixed rapidly with different concentrations of ATP and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Two kinetic phases were observed and assigned by (i) analyzing the dependence of the corresponding observed rate constants on ATP concentration; and (ii) by carrying out mixing experiments also with ADP, ATPgammaS and ATP without K(+). The values of the observed rate constants corresponding to both phases are found to be dependent on ATP concentration. The observed rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are similar to those determined in steady-state ATPase experiments. This phase most likely reflects ATP binding-induced conformational changes. The rate constant of the conformational change in the presence of excess ATP is about 17s(-1) (at 25 degrees C) and is tenfold slower than the corresponding rate constant of GroEL. The observed rate constant corresponding to the second slower phase displays a hyperbolic dependence on ATP concentration. This phase is not observed in mixing experiments of CCT with ADP, ATPgammaS or ATP without K(+) and it, therefore, reflects a conformational change associated with ATP hydrolysis. Taken together, our results indicate that the kinetic mechanism of the allosteric transitions of CCT differs considerably from that of GroEL.
Kafri
Galit
G
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
Horovitz
Amnon
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013737:Testis / Q000378:metabolism; D013997:Time Factors
D000255:Adenosine Triphosphate; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins