(Received for publication, June 25, 1994; and in revised form, November 2, 1994)
From the
Several functions of the 70-kilodalton heat shock cognate
protein (Hsc70), such as peptide binding/release and clathrin
uncoating, have been shown to require potassium ions. We have examined
the effect of monovalent ions on the ATPase activity of Hsc70. The
steady-state ATPase activities of Hsc70 and its amino-terminal 44-kDa
ATPase fragment are minimal in the absence of K and
reach a maximum at
0.1 M [K
].
Activation of the ATPase turnover correlates with the ionic radii of
monovalent ions; those that are at least 0.3 Å smaller
(Na
and Li
) or larger
(Cs
) than K
show negligible
activation, whereas ions with radii differing only
0.1 Å
from that of K
(NH
and
Rb
) activate to approximately half the turnover rate
observed with K
. Single turnover experiments with
Hsc70 demonstrate that ATP hydrolysis is 5-fold slower with
Na
than with K
. The equilibrium
binding of ADP or ATP to Hsc70 is unperturbed when K
is replaced with Na
. These results are
consistent with a role for monovalent ions as specific cofactors in the
enzymatic hydrolysis of ATP.
The 70-kDa heat shock protein family is a highly conserved,
ubiquitous group of proteins believed to function as
``chaperones,'' facilitating folding, assembly, or
disassembly of other proteins but not forming part of the finished
product (for reviews, see (1, 2, 3, 4) ). All members of the
family have an ATPase activity, which regulates cycles of binding and
release from unfolded proteins, although the exact mechanism is still
unclear. Hsc70 ()is a constitutively expressed mammalian
member of this family; it was originally described as a
clathrin-uncoating protein(5) .
The Hsc70 protein can be divided into several functional domains: an amino-terminal 44-kDa ATPase domain, an 18-kDa peptide binding domain, and a carboxyl-terminal 10-kDa domain of unknown function(6, 7) . The crystal structure of the 44-kDa ATPase domain has been determined(8) . The tertiary fold is similar to that of actin, which is also an ATPase. Furthermore, the structures of the ATP binding domains of hexokinase and glycerol kinase, two other phosphotransferases, are similar to those of actin and the Hsc70 ATPase fragment(9) .
There have been several
reports that monovalent cations affect activities of Hsc70. Recently,
it was shown that both K and ATP are required for the
dissociation of denatured proteins from DnaK (the Escherichia coli Hsc70 homolog) and that Na
will not substitute
for K
(10) . Potassium ion is required for in vitro reconstitution of a complex of Hsc70, steroid
receptor, and Hsp90(11) . In this case,
NH
or Rb
cations
could substitute for K
, but Na
and
Li
could not. In the original biochemical
characterization of the clathrin uncoating activity of Hsc70, it was
noted that K
or NH
above a threshold concentration of approximately 10-20
mM was required for uncoating activity(12) .
All of these reports describe functions of Hsc70 that involve a combination of polypeptide binding, ATPase activity, and a coupling between them; they do not indicate which specific activity is dependent on monovalent cation. In this context, we have examined the question of whether the ATPase activity of Hsc70, in particular, is dependent on monovalent cations and have concluded that both the rate of ATP hydrolysis and the rates of binding and release of nucleotide are affected by the type and concentration of ion. The characteristics of the stimulation of the Hsc70 ATPase activity by monovalent cations are similar in ion specificity and concentration dependence to those displayed by a number of enzymes, including many phosphotransferases. The direct participation of monovalent ions as cofactors in the ATPase activity of Hsc70, which we postulate from the kinetic and structural results presented here and in the accompanying manuscript(28) , may represent a phenomenon that is an essential feature of a significant number of phosphotransferase reactions.
Single turnover
rates of ATP hydrolysis were determined at 37 °C in 36-µl
reaction mixtures containing 10 mM HEPES, pH 7.0, 4.5 mM magnesium acetate, 4.3 µCi of
[-
P]ATP (
40 nM), 100 mM KCl or NaCl, and an excess of Hsc70 (2 or 10 µM, as
noted). Aliquots of the reaction were stopped at specific times by
addition of an equal volume of ice-cold 12% trichloroacetic acid,
followed rapidly by neutralization with 10 mM HEPES, pH 7.0,
0.4 M KOH. 0.5 µl of each stopped reaction was spotted
onto a polyethyleneimine-cellulose TLC plate; TLC was performed and
product ADP determined as above.
The rate of association of
nucleotide-free Hsc70 and labeled ATP was assayed at 37 °C in 10
mM HEPES, pH 7.0, 4.5 mM magnesium acetate, and 100
mM KCl or NaCl. The binding reactions were initiated by mixing
equal volumes of prewarmed solutions of 2 enzyme and 2
buffer + 2 nM [
-
P]ATP in a
reaction volume of 100 µl. At the indicated time points, 10-µl
aliquots of the solution were spotted onto BA85 nitrocellulose membrane
filters (Schleicher and Schuell) and washed with 1-2-ml reaction
buffer. For each zero time point, 10 µl of ATP solution with no
protein present was spotted onto a filter and washed as above. Filters
were dried and Cerenkov radiation was counted in a Beckman LS 5000TA
scintillation counter. The apparent association rate k
was computed by fitting a single exponential of the form P(1 - e
obs
) to
the fraction of background-corrected counts retained on filters versus time, where P is the asymptotic value of the
function.
To determine equilibrium binding constants (K) for ADP, [
-
P]ADP,
prepared as described, (
)was mixed with Hsc70 ranging in
concentration 0.125-8.0 µM in the buffers described
above. The solution was filtered, and K
was
determined by a least squares fit of the
function,
to the data, where is the fraction of counts retained on
the filter and
is the asymptotic value at high
[Hsc70], which is the filter efficiency.
The ATPase cycle for Hsc70 and its 44 kDa ATPase domain can be described by the following scheme.
where ATP hydrolysis is essentially irreversible (k
0) and product release is
ordered.
We use this scheme to interpret the results
presented below.
Figure 1:
Effect of increasing monovalent cation
concentration on ATP hydrolysis by Hsc70. The error bars represent the residual error in the slope of a straight line fit
to these time points; they should be regarded as minimum estimates of
error. A, full-length Hsc70 with NaCl (), NaCl + 2
mM VHLTPVGK (
), KCl (
), or KCl + 2 mM VHLTPVGK (
). B, 44-kDa ATPase fragment of Hsc70
with NaCl (
) or KCl (
).
The ATPase activity of full-length Hsc70 can be stimulated in vitro 2-3-fold by addition of denatured protein (18) or short peptides(19) . When 2 mM of a peptide known to stimulate ATPase activity (VHLTPVGK, Sigma) was added to the reactions, all hydrolysis rates were increased approximately 2-fold (Fig. 1A). A marked preference for KCl over NaCl was again observed.
We next tested the effect of monovalent cations on the steady-state ATPase activity of the 44-kDa ATPase fragment of Hsc70 (Fig. 1B). KCl was preferred to NaCl at all concentrations, and the observed rates in the presence of NaCl did not increase significantly with increasing NaCl. The apparent hydrolysis rate reached a maximum at 250 mM KCl; at salt concentrations above this, distortion of the TLC separations by the salt made quantitation of results unreliable.
LiCl, NHCl, RbCl,
and CsCl were also tested for their ability to stimulate the
steady-state ATPase activity of Hsc70. Data for Hsc70 ATPase activities
in the presence of 150 mM monovalent cation are shown in Fig. 2; the activities showed the same order of cation
preference at concentrations of 75 mM and 225 mM as
well (data not shown). K
stands out as the monovalent
cation that is most effective in stimulating the ATPase activity of
Hsc70. NH
and Rb
,
with ionic radii differing less than 0.2 Å from that of
K
, are partially effective, whereas cations whose
ionic radii differ from that of K
by > 0.3 Å (either larger or smaller) are ineffective in stimulating the ATPase
activity.
Figure 2: Rate of ATP hydrolysis by Hsc70 in the presence of various monovalent cations (chloride salts at 150 mM). Ionic radii are from (27) .
Figure 3:
Binding kinetics of ATP and Hsc70 in the
presence of 100 mM KCl (A, B) or 100 mM NaCl (C, D). Experiments were performed as described under
``Materials and Methods.'' Enzyme concentrations were 20
nM (), 40 nM (
), 60 nM (
),
80 nM (
) (A); 50 nM (
), 100
nM (
), 200 nM (
), 400 nM (
) (C). k
values, determined
from exponential fits as described in the text, were plotted against
enzyme concentration and fit to a linear equation to determine kinetic
constants (B, D).
Figure 4:
Equilibrium binding of
[-
P]ADP to Hsc70 in the presence of 100
mM KCl (
) or 100 mM NaCl (
). Curves show
the result of a least squares fit of a hyperbolic function to the
data.
Data showing the time course of ADP
formation under single turnover conditions are shown in Fig. 5.
The rate of hydrolysis is substantially slower in the presence of 100
mM NaCl than in the presence of 100 mM KCl. The
observed rate of ATP hydrolysis will reach a plateau as the enzyme
concentration exceeds the K. The K
for ATP in the presence of KCl is known to be about 0.5
µM(13, 14) , but the K
in the presence of NaCl has not been determined. We therefore
tested enzyme concentrations of 2 µM and 10 µM in the presence of NaCl; the data points fall on essentially the
same curve, and therefore the observed hydrolysis rate has reached a
plateau. Values of k
determined from fits to the
single exponential equation are 0.013 ± 0.002 s
in 100 mM KCl, and 0.0023 ± 0.0004 s
in 100 mM NaCl (average of 2 and 10 µM determinations).
Figure 5: Single turnover kinetics of Hsc70 in the presence of 100 mM KCl or NaCl. The amount of ATP hydrolysis at the indicated times was determined as described under ``Materials and Methods,'' with reactions containing 40 nM ATP and an excess of enzyme as noted.
We have demonstrated that the steady-state ATPase activity of
Hsc70 and its 44-kDa ATPase fragment are stimulated by monovalent
cation. The maximum ATPase turnover rate varies with the size, or ionic
radius, of the cation, with K giving the highest
activity and ions that are either smaller or larger giving lower
activity. Since Hsc70 and the 44-kDa ATPase domain show similar
dependence on monovalent ions, the effect on ATPase activity can be
decoupled from peptide binding and the peptide binding domain.
Comparison of pre-steady-state measurements of ATP binding and
hydrolysis shows that both the hydrolysis rate and the binding and
release rates are much slower in the presence of Naversus K
. Also, the steady-state ATPase
rate is 10-fold slower in the presence of Na
compared
with K
. However, the equilibrium dissociation
constants for the Hsc70
-ADP complex measured in the presence of
Na
versus K
are equal within
experimental error, as are the dissociation constants computed from the
ATP association rates for the Hsc70
-ATP complex. Thus,
substitution of Na
for K
affects the
kinetics of several steps of the ATPase cycle, but does not
substantially alter the affinity for nucleotides.
The concentration
of K required for optimal Hsc70 ATPase activity,
0.1 M, is high compared with, for example, the
concentration of Mg
that gives optimal activity,
10 µM(13) . It is worth noting in this
context that the binding constants for K
are
anticipated to be substantially weaker than those for
Mg
, since the electrostatic interactions of
monovalent ions with their ligands are much weaker than those of
divalent ions. Also, the cytoplasmic concentration of K
is 0.1-0.2 M, so that the protein would be
maximally active under physiological conditions. The weaker binding
affinity of monovalent ions is expected on energetic grounds and does
not imply that their interactions with enzymes are less specific than
those of divalent ions; on the contrary, in cases where it has been
characterized structurally, their binding is very
specific(21, 22) , also see accompanying
manuscript(28) .
Several dozen enzymes are known to require
monovalent cations for their activity(23) . Many of them are
phosphotransferases, including a few ATPases. Several patterns of
response to monovalent cations are seen among these
enzymes(24) . One common pattern is that K (optimal concentration range typically 0.01-0.20 M) gives the greatest activation,
NH
and Rb
give lesser
activation, and Na
and Li
fail to
activate. The behavior of Hsc70 is consistent with this pattern.
Recently, another example in which K
is required to
optimize enzymatic ATP hydrolysis has been reported. The E. coli GroEL protein, a molecular chaperone of the Hsp60 family, is fully
active in both ribulosebisphosphate carboxylase folding and uncoupled
ATP hydrolysis in the presence of K
,
Rb
, and NH
; however,
it is essentially inactive in the absence of monovalent cation or in
the presence of Na
, Li
, or
Cs
(25, 26) .
One general question
that arises is whether monovalent ions act as allosteric effectors,
influencing the overall structure of an enzyme, or whether they
interact directly with substrates. Dialkylglycine decarboxylase
provides a precedent for the former mode of interaction; K binding at a specific site remote from substrates activates the
enzyme, whereas Na
binding at the same site inhibits
it(22) . The crystallographic observation that two K
ions bind in the active site region of the ATPase domain of
Hsc70, at sites where they could act as metal cofactors in the ATP
hydrolysis reaction(28) , provides an example of the latter
mode, activation through direct interaction with substrate. The
activities of a substantial number of phosphotransferase enzymes show a
pattern of dependence on monovalent ion that parallels what we observe
with Hsc70. This suggests that direct participation of K
as a metal cofactor, by interaction with the nucleotide
substrate, may be a relatively common mechanism by which monovalent
ions influence phosphotransferase reactions.