Switches, Catapults, and Chaperones: Steady-State Kinetic Analysis of Hsp70-Substrate Interactions
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- 作者：Liudmila S. Chesnokova and Stephan N. Witt
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：August 23, 2005
- 年：2005
- 卷：44
- 期：33
- 页码：11224 - 11233
- 全文大小：161K
- 年卷期：v.44,no.33(August 23, 2005)
- ISSN：1520-4995
文摘
Hsp70 chaperones are heterotropic allosteric systems in which ATP and misfolded or aggregatedpolypeptides are the activating ligands. To gain insight into the mechanism by which ATP and polypeptidesregulate Hsp70 chaperone activity, the effect of a short peptide on the KM for ATP was analyzed usingthe Escherichia coli Hsp70 called DnaK. In the absence of peptide, the 
qn/bi050787be10001.gif"> for ATP is 52 ± 11 nM,whereas this value jumps to 14.6 ± 1.6 
M in the presence of saturating peptide. This finding supportsa mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes thechaperone back in the opposite direction (and thus increases KM), according to ATP + DnaK·P 
ATP·DnaK·P ATP·DnaK* + P, where ATP·DnaK·P is an intermediate from which competing ATP hydrolysisoccurs (ATP·DnaK·P 
ADP·DnaK·P). We show that this branched mechanism can even explain howDnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATPhydrolysis (khy) in the absence of peptide may be as high as 0.5 s-1 (rather than 5 × 10-4 s-1 as oftenstated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysisand effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in thefollowing relation: kcat = khy/1000 = 5 × 10-4 s-1. How polypeptide substrates and the co-chaperoneDnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5s-1, is discussed.
M in the presence of saturating peptide. This finding supportsa mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes thechaperone back in the opposite direction (and thus increases KM), according to ATP + DnaK·P ATP·DnaK·P ATP·DnaK* + P, where ATP·DnaK·P is an intermediate from which competing ATP hydrolysisoccurs (ATP·DnaK·P ADP·DnaK·P). We show that this branched mechanism can even explain howDnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATPhydrolysis (khy) in the absence of peptide may be as high as 0.5 s-1 (rather than 5 × 10-4 s-1 as oftenstated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysisand effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in thefollowing relation: kcat = khy/1000 = 5 × 10-4 s-1. How polypeptide substrates and the co-chaperoneDnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5s-1, is discussed.