Transient Kinetic Analysis of ATP Hydrolysis by the CCT/TRiC Chaperonin.
The chaperonin-containing t-complex polypeptide 1 (CCT, also known as TRiC) assists protein folding in an ATP-dependent manner. CCT/TRiC was mixed rapidly with different concentrations of ATP, and the amount of phosphate formed upon ATP hydrolysis was measured as a function of time using the coumarin-labeled phosphate-binding protein method. Two burst phases were observed, followed by a lag phase and then a linear steady-state phase of ATP hydrolysis. The phases were assigned by (i) determining their dependence on ATP and K+concentrations and (ii) by measuring their sensitivity to the mutation Gly345→Asp in subunit CCT4, which decreases cooperativity in ATP binding. The values of the observed rate constants corresponding to the burst phases are found to decrease with increasing ATP and K+concentrations, thereby indicating that the apo state of CCT/TRiC is in equilibrium between several conformations and that "conformational selection" by ATP takes place before hydrolysis. The amplitude of the lag phase, which follows, decreases with increasing ATP concentrations, thus indicating that it reflects a transition between states with low affinity for ATP and a state with high affinity for ATP that is predominant under steady-state conditions. A kinetic model based on the data is suggested, in which CCT/TRiC is in equilibrium between a relatively large number of states that are distinguished kinetically, in agreement with its proposed sequential allosteric mechanism.
Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
Ilia
I
Korobko
Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
Michal
M
Nadler-Holly
Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: Amnon.Horovitz@weizmann.ac.il.
Amnon
A
Horovitz
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D056404:Chaperonin Containing TCP-1 / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011188:Potassium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C527547:CCT4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D056404:Chaperonin Containing TCP-1; D011188:Potassium
ATP hydrolysis; allostery; chaperonins; transient kinetics