(Received for publication, November 27, 1995; and in revised form, December 29, 1995)
From the
Cys-674 of the sarcoplasmic reticulum
Ca-ATPase was labeled with N-acetyl-N`-(5-sulfo-1-naphthyl)ethylenediamine
without a loss of the catalytic activity. The ATP-induced drop in the
fluorescence of the label, which was shown in our previous studies to
reflect the conformational change upon formation of the
calcium
enzyme
ATP complex, was followed by the stopped-flow
method. The subsequent phosphoenzyme formation was followed by the
rapid quenching method. Effects of a partial substitution of organic
solvents for water in the medium on the conformational change and
phosphoenzyme formation were investigated in the presence of 100
µM CaCl
at pH 7.5, 0 °C. The rate of the
conformational change increased with increasing ATP concentration
(0.1-100 µM) and was unaffected by 30% (v/v)
dimethyl sulfoxide. In contrast, the rate of phosphoenzyme formation
decreased sharply with increasing concentration of dimethyl sulfoxide
(20-40% (v/v)), even when phosphoenzyme formation was saturated
with ATP. N,N-Dimethylformamide and glycerol had essentially
the same effects as dimethyl sulfoxide. These results show that the
reduction in water activity does not affect the rate of the
conformational change upon formation of the
calcium
enzyme
ATP complex, but greatly retards the
subsequent phosphoryl transfer from ATP to the enzyme protein. This
strongly suggests that in this early stage of the catalytic cycle water
plays a critical role in ensuring the rapid turnover of the enzyme.
The SR ()Ca
-ATPase catalyzes ATP
hydrolysis coupled to Ca
transport(1, 2) . In the initial step of the
catalytic cycle, the enzyme is activated through Ca
binding to the high affinity transport site. The
-phosphoryl
group of ATP is transferred to Asp-351 in the catalytic site of the
activated enzyme (3, 4, 5, 6) to
form ADP-sensitive EP(7, 8, 9) . In
the subsequent conformational transition, this EP is converted
to ADP-insensitive form. Concurrently, the affinity of the transport
site for Ca
is greatly reduced, and the
Ca
is released into the lumen. Finally,
ADP-insensitive EP is hydrolyzed to liberate P
.
This catalytic cycle is fully reversible.
The and Hasselbach (10) showed previously that the rate of EP hydrolysis
is markedly reduced by MeSO. Later, de Meis et al. (11, 12, 13) found that EP formation
from P
in the reverse reaction is greatly favored by
substituting organic solvents such as Me
SO, DMF, and
glycerol for water in the medium. They further suggested that, in
contrast to the catalytic site of ADP-insensitive EP, which is
thought to be hydrophobic(11) , the catalytic site of
ADP-sensitive EP is hydrophilic. According to this proposal, a
change in water activity within the catalytic site may be essential for
the conformational transition from ADP-sensitive EP to
ADP-insensitive EP(11, 14, 15) .
However, the role of water in the formation of ADP-sensitive EP from ATP in the early stage of the catalytic cycle has not
yet been explored.
Previously, we labeled Cys-674 of the enzyme by EDANS selectively without a loss of the catalytic activity and found that the fluorescence of bound EDANS decreases greatly upon formation of the calcium-enzyme-substrate complex in the initial step of the catalytic cycle(16, 17, 18) . This fluorescence drop reflects a conformational change in the vicinity of the ATP-binding site, because Cys-674 is surrounded by amino acid residues which contribute the conformation of the ATP-binding site(19, 20, 21, 22, 23, 24, 25) . This conformational change is immediately followed by the phosphoryl transfer from ATP to the enzyme protein (16, 17, 18) .
In this study, we have
investigated the effects of organic solvents (MeSO, DMF,
and glycerol) on the conformational change in the
calcium-enzyme-substrate complex and EP formation from ATP by
using EDANS-labeled SR vesicles. The conformational change has been
followed by the stopped-flow measurements of the fluorescence of bound
EDANS and EP formation followed by the continuous-flow rapid
quenching method. The results demonstrate that the reduction in water
activity by addition of the organic solvents does not appreciably
affect the rate of the conformational change, but greatly retards the
subsequent phosphoryl transfer. These findings suggest that in this
early stage of the catalytic cycle water plays a critical role in
ensuring the rapid turnover of the enzyme.
Figure 1:
Effects of MeSO, DMF, and
glycerol on the kinetics of ATP-induced fluorescence drop.
Ca
-loaded, EDANS-labeled SR vesicles (0.05 mg/ml) in
a medium containing 10 mM MgCl
, 0.1 mM CaCl
, 0.1 M KCl, and 20 mM Tris/HCl
(pH 7.5) in the absence of organic solvents (A) or in the
presence of 30% (v/v) Me
SO (B), 30% (v/v) DMF (C), or 30% (v/v) glycerol (D) were mixed with ATP in
the same medium at 0 °C by the stopped-flow method. ATP
concentrations after the mixing are indicated in the
figure.
Figure 2:
Effect of MeSO on the kinetics
of EP formation. EP formation was performed with
[
-
P]ATP in the presence of different
concentrations of Me
SO by the continuous-flow rapid
quenching method under the otherwise same conditions as in Fig. 1. Me
SO concentrations used were 0% (
),
20% (
), 30% (
), and 40% (
,
) (v/v).
[
-
P]ATP concentrations after the mixing
were 100 (
,
,
,
) and 300 (
)
µM. For comparison, the ATP-induced fluorescence change
(
) determined as in Fig. 1in the presence
of 30% (v/v) Me
SO is shown after subtraction of the
base-line level, which was determined by mixing the vesicles with the
ATP-free medium.
In order to see
whether the observed effect of MeSO on EP
formation is due to reduction in the water activity or it is specific
to this organic solvent, effects of two other organic solvents, DMF and
glycerol, were investigated in the same way as described in Fig. 2except that DMF or glycerol was used in place of
Me
SO. A similar retardation of EP formation was
found with either DMF (Fig. 3) or glycerol (Fig. 4),
although glycerol was somewhat less effective than Me
SO and
DMF. In fact, the rate of EP formation decreased 20-fold by
40% (v/v) DMF and 13-fold by 40% (v/v) glycerol. An increase in the ATP
concentration from 100 µM to 300 µM again
gave no increase in the rate of EP formation which was
retarded by 40% (v/v) DMF or glycerol. The steady-state level of EP was unaffected by these organic solvents, being 3.65
nmol/mg at 40 s in the presence of 40% (v/v) DMF and 4.05 nmol/mg at 30
s in the presence of 40% (v/v) glycerol (not shown). The EDANS
fluorescence drop in the presence of 30% (v/v) DMF or glycerol was much
faster than EP formation under the same conditions. These
results are essentially the same as those obtained with
Me
SO, strongly suggesting that the observed retardation of EP formation is due to the reduction in water activity. It is
evident that the observed effects of the organic solvents are not due
to changes in the dielectric constant of the medium, because the
dielectric constant decreases considerably upon addition of DMF or
glycerol but only slightly upon addition of Me
SO. Indeed,
the dielectric constants of water, 40% (v/v) Me
SO, 40%
(v/v) DMF, and 40% (v/v) glycerol at 0 °C are 88.1, 85.1, 77.1, and
75.7, respectively(29) .
Figure 3:
Effect of DMF on the kinetics of EP formation. EP formation was performed as in Fig. 2except that MeSO was replaced by DMF. DMF
concentrations used were 0% (
), 20% (
), 30% (
), and
40% (
,
) (v/v). [
-
P]ATP
concentrations after the mixing were 100 (
,
,
,
) and 300 (
) µM. For comparison, the
ATP-induced fluorescence change (
) was determined
in the presence of 30% (v/v) DMF, otherwise same as described in the
legend to Fig. 2.
Figure 4:
Effect of glycerol on the kinetics of EP formation. EP formation was performed as in Fig. 2except that MeSO was replaced by glycerol.
Glycerol concentrations used were 0% (
), 20% (
), 30%
(
), and 40% (
,
) (v/v).
[
-
P]ATP concentrations after the mixing
were 100 (
,
,
,
) and 300 (
)
µM. For comparison, the ATP-induced fluorescence change
(
) was determined in the presence of 30% (v/v)
glycerol, otherwise same as described in the legend to Fig. 2.
Figure 5:
Dependence of the extent of fluorescence
drop on concentrations of AMP-PCP and ADP. A, the extent of
the AMP-PCP- or ADP-induced fluorescence drop attained in the steady
state was determined at 0 °C by adding small volumes of AMP-PCP
(,
) or ADP (
,
) to 2.4 ml of a suspension of
EDANS-labeled SR vesicles (0.025 mg/ml) in a medium containing 10
mM MgCl
, 0.1 M KCl, 20 mM Tris/HCl (pH 7.5), and 0.1 mM CaCl
in the
absence (
,
) or presence (
,
) of 40% (v/v)
Me
SO. In all the measurements, the total volumes of added
AMP-PCP or ADP were less than 0.05 ml. The fluorescence drop was
corrected for dilution upon each addition. The data in the low AMP-PCP
or ADP concentration range in upper panel were replotted in lower panel. B, the measurements were performed in the
presence of 5 mM EGTA in place of CaCl
, otherwise
same as described in A. The symbols correspond to those in A. The solid lines in A and B show
least squares fit to a Michaelis equation.
When AMP-PCP was added in the presence of Ca and
absence of organic solvents, the fluorescence decreased with increasing
concentration of AMP-PCP (Fig. 5A). The K
value obtained is consistent with the previously
reported affinity of the catalytic site of the enzyme for
AMP-PCP(30, 31) . This is in accord with our previous
conclusion (17) that the fluorescence drop reflects a
conformational change occurring upon formation of the
calcium-enzyme-substrate complex. When Me
SO was added to
give 40% (v/v), the affinity of the enzyme for AMP-PCP increased
greatly, and the K
became 50-fold lower than that
in the absence of organic solvents. The K
value
for ADP obtained in the presence of Ca
and absence of
organic solvents is also consistent with the previously reported
affinity of the catalytic site for ADP (32) . When
Me
SO was added to give 40% (v/v), the K
for ADP again decreased greatly. Similar results were obtained
with DMF, although DMF was less effective than Me
SO. On the
other hand, 40% (v/v) glycerol caused no appreciable decrease in the K
for AMP-PCP and ADP.
The K for AMP-PCP and ADP in the absence of Ca
decreased only slightly when Me
SO was added to give
40% (v/v) and rather increased to some extent when DMF or glycerol was
added to give 40% (v/v) (Fig. 5B and Table 1).
These results are in contrast to those obtained in the presence of
Ca
. Accordingly, the conformational change
responsible for the ATP (or its analog)-induced fluorescence drop in
the presence of Ca
is distinct from that in the
absence of Ca
. It is, therefore, very likely that the
observed effects of Me
SO and DMF on the affinity for
AMP-PCP or ADP in the presence of Ca
are specific to
the Ca
-activated enzyme.
The mechanism of the phosphoryl transfer retardation induced by the reduction in water activity and a possible role of water in the early stage of the catalytic cycle may be conveniently discussed in terms of the following reaction scheme proposed previously (16, 17, 18) ,
where E, E`, and E" denote different
conformational states of the Ca-activated enzyme, and
S denotes the substrate (ATP or its analog). E"P represents
ADP-sensitive EP. Our previous
findings(16, 17, 18) revealed that most of
the ATP-induced fluorescence drop (Fig. 1), as well as the whole
of the fluorescence drop induced by nonhydrolyzable ATP analogs (Fig. 5) in the presence of Ca
, occurs upon
the conformational change in Step 1. The present results (Fig. 2Fig. 3Fig. 4) show that the reduction in
water activity markedly increases the activation energy for the
phosphoryl transfer in Step 2. This strongly suggests that in this
early stage of the catalytic cycle water plays a critical role in
ensuring the rapid turnover of the enzyme, although the stereochemical
analysis of the phosphoryl transfer in the SR
Ca
-ATPase (33) previously presented
convincing evidence for the in-line displacement mechanism of the
phosphoryl transfer in which water molecules are not directly involved.
The increase in the activation energy for the phosphoryl transfer by
the reduction in water activity may be possibly due to unstabilization
of the transition state in Step 2. This suggests that the transition
state is stabilized by hydration when the water activity has not been
reduced. The data (Table 1) showing the lack of the
glycerol-induced shift of the equilibrium between
CaE + S and
Ca
E`
S imply that the energy levels of
both Ca
E + S and
Ca
E`
S are equally raised, rather than
equally lowered, by the addition of glycerol, because glycerol is less
hydrophilic than water and thus unstabilizes hydrated substrates such
as ATP and its analogs (cf.(11) ). This gives a
support to the above possibility that the observed increase in the
activation energy for the phosphoryl transfer is due to unstabilization
of the transition state in Step 2 rather than stabilization of
Ca
E`
S. However, it should be noted
that the suggested hydration of the transition state is seemingly out
of harmony with the well known tight binding of transition state
analogs to amino acid residues within the catalytic
site(34, 35, 36) .
The results (Fig. 1) show that the addition of any of the three organic
solvents causes no change or only a slight decrease in the rate of the
conformational change in Step 1. This finding indicates that the energy
levels of CaE + S and the transition state in Step
1 are almost equally raised by the reduction in water activity. In
contrast, the Me
SO- or DMF-induced large reductions in the K
for formation of
Ca
E`
S (Table 1) indicate that
the rise in the energy level of Ca
E`
S
comparable with that in the energy level of Ca
E + S is not induced by the addition of Me
SO and
DMF. It is, therefore, likely that the energy level of
Ca
E`
S is less sensitive to these two
considerably hydrophobic solvents than that of
Ca
E + S. This is consistent with the
possible hydrophobic tertiary structure of the ATP-binding site, which
was proposed previously by Taylor and Green (37) on the basis
of the predicted secondary structure of the enzyme. Although the reason
why the effect of glycerol on the relative energy levels of
Ca
E + S and
Ca
E`
S is different from those of
Me
SO and DMF remains obscure, this difference might be
possibly due to the fact that glycerol is less hydrophobic than
Me
SO and DMF(11) .