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J Biol Chem, Vol. 275, Issue 11, 7779-7786, March 17, 2000
From the Department of Physiology and Biophysics, University of
California, Irvine, California 92697
Hsc66 from Escherichia coli is a
constitutively expressed hsp70 class molecular chaperone whose activity
is coupled to ATP binding and hydrolysis. To better understand the
mechanism and regulation of Hsc66, we investigated the kinetics of ATP
hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state
experiments revealed that Hsc66 has a low affinity for ATP
(KmATP = 12.7 µM) compared with other hsp70 chaperones. The kinetics of
nucleotide binding were determined by analyzing changes in the Hsc66
absorbance spectrum using stopped-flow methods at 23 °C. ATP binding
results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm;
this is interpreted as arising from a two-step process in which ATP
binding (kaATP = 4.2 × 104 M
Kinetic Characterization of the ATPase Cycle of the Molecular
Chaperone Hsc66 from Escherichia coli*
1
s1, kdATP = 1.1 s1) is followed by a slow conformational
change (kconf = 0.1 s1). Under single
turnover conditions, the ATP-induced transition decays exponentially
with a rate (kdecay = 0.0013 s1) similar to that observed in both steady-state and
single turnover ATP hydrolysis experiments
(khyd = 0.0014 s1). ADP
binding to Hsc66 results in a monophasic transition in the absence
(kaADP = 7 × 105 M1 s1,
kdADP = 60 s1) and presence of physiological levels of inorganic
phosphate (kaADP(Pi)) = 0.28 × 105 M1
s1,
kdADP(Pi) = 9.1 s1). These results indicate that ATP
hydrolysis is the rate-limiting step under steady-state conditions and
is >103-fold slower than the rate of ADP/ATP exchange.
Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been
characterized to date, the R 
T equilibrium balance for Hsc66 is
shifted in favor of the low peptide affinity T state, and regulation of
the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.
*
This work was supported by National Institutes of Health
Grant GM54624 and Training Grant GM07311.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, University of California, Irvine, CA 92697. Tel.:
949-824-6580; Fax: 949-824-8540; E-mail: lvickery@uci.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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