posted on 1998-01-13, 00:00authored byRick Russell, Robert Jordan, Roger McMacken
DnaK, the prototype Hsp70 protein of Escherichiacoli, functions as a molecular chaperone
in protein folding and protein disassembly reactions through cycles of
polypeptide binding and release
that are coupled to its intrinsic ATPase activity. To further our
understanding of these processes, we
sought to obtain a quantitative description of the basic ATPase cycle
of DnaK. To this end, we have
performed steady-state and pre-steady-state kinetics experiments and
have determined rate constants
corresponding to individual steps in the DnaK ATPase cycle at 25 °C.
Hydrolysis of ATP proceeds very
slowly with a rate constant (khyd ≈ 0.02
min-1) at least 10-fold smaller than the rate
constant for any
other first-order step in the forward reaction pathway. The ATP
hydrolysis step has an activation energy
of 26.2 ± 0.4 kcal/mol and is rate limiting in the steady-state under
typical invitro conditions.
ATP
binds with unusual strength to DnaK, with a measured
KD ≈ 1 nM. ADP binds considerably less
tightly
than ATP and dissociates from DnaK with a koff
of ∼0.4 min-1 (compared with a
koff of ∼0.008
min-1
for ATP). However, in the presence of physiologically relevant
concentrations of inorganic phosphate
(Pi), the release of ADP from DnaK is greatly slowed,
approximately to the rate of ATP hydrolysis.
Under these conditions, the ADP-bound form of DnaK, the form that
binds substrate polypeptides most
tightly, was found to represent a significant fraction of the DnaK
population. The slowing of ADP release
by exogenous Pi is due to thermodynamic coupling of the
binding of the two ligands, which produces a
coupling energy of ∼1.6 kcal/mol. This result implies that
product release is not strictly ordered. In the
absence of exogenous inorganic phosphate, Pi product, by
virtue of its higher koff, is released prior
to
ADP. However, at physiological concentrations of inorganic
phosphate, the alternate product release
pathway, whereby ADP dissociates from a ternary
DnaK·ADP·Pi complex, becomes more
prominent.