(Received for publication, June 30, 1994; and in revised form, September 19, 1994)
From the
The roles of the hydrophobic side chains of residues
Phe, Ile
, Tyr
,
Leu
, and Ile
located at the M5S5 boundary
of the Ca
-ATPase of sarcoplasmic reticulum were
analyzed by site-directed mutagenesis. Substitution of Tyr
with glycine resulted in a new phenotypic variant of the
Ca
-ATPase that catalyzed a high rate of
Ca
-activated ATP hydrolysis without net accumulation
of Ca
in the microsomal vesicles. The ATPase activity
of the Tyr
Gly mutant displayed characteristics
similar to the ATPase activity of the wild-type enzyme measured in the
presence of calcium ionophore, and the mutant was able to form the
ADP-insensitive phosphoenzyme intermediate. Mutants Phe
Gly, Ile
Gly, Leu
Gly, and Ile
Gly were able to
accumulate Ca
. In mutants Leu
Gly and Ile
Gly, the turnover rate was low due to
inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme
intermediate. On the other hand, mutant Leu
Lys
dephosphorylated rapidly. Mutants Phe
Gly and
Leu
Lys displayed apparent Ca
affinities that were reduced two and three orders of magnitude,
respectively, relative to that of the wild-type.
The Ca-ATPase of sarcoplasmic reticulum
belongs to the ion-translocating ATPases of P-type in which the
hydrolysis of ATP is coupled to vectorial transport through formation
of an acid-stable phosphoryl aspartyl enzyme intermediate that exists
in two major conformational states E1P (
)and E2P (De Meis, 1981; Andersen, 1989). Analysis of the amino
acid sequence (MacLennan et al., 1985) along with extensive
site-directed mutagenesis studies (MacLennan et al., 1992;
Andersen and Vilsen, 1992a) and studies of two-dimensional membrane
crystals (Taylor et al., 1986; Toyoshima et al.,
1993) have concurred in a structural model for the
Ca
-ATPase in which the Ca
binding
region is located within a cluster of putative transmembrane
-helices, while the catalytic ATP-binding site is made up from
residues in the cytoplasmic domains (Fig. 1). The presence in
the Ca
-ATPase of distinct structural entities
associated with ATP binding and Ca
binding,
respectively, has recently been confirmed by studies of chimeric fusion
proteins (Sumbilla et al., 1993; Nørregaard et
al., 1993). Hence, the energy coupling between scalar and
vectorial reactions seems to require long distance communication
between the cytoplasmic and transmembrane parts of the pump through
conformational changes (Andersen and Vilsen, 1992a). The mutational
analysis of the Ca
-ATPase has pinpointed the three
transmembrane segments M4, M5, and M6 as central to events associated
with Ca
binding and occlusion (Clarke et
al., 1989; Andersen and Vilsen, 1992b, 1994; Vilsen and Andersen,
1992d). In addition, this region may be involved in the
countertransport of protons (Andersen and Vilsen 1992b, 1994; Vilsen
and Andersen, 1992c). An important task is also to locate the amino
acid residues responsible for maintenance of the normal physiological
coupling of ATP hydrolysis to ion transport, but so far no
site-directed mutagenesis data have revealed uncoupled mutants of the
Ca
-ATPase.
Figure 1:
Structural model of the
Ca-ATPase highlighting the residues at the M5S5
boundary. This model is based on the structure prediction by MacLennan et al.(1985) and Green(1989). S and M indicate stalk and transmembrane helices, respectively. Each circle corresponds to an amino acid residue indicated by the one-letter code inside the circle. The residues at the M5S5
boundary are shown enlarged in a box. Residues with
oxygen-containing side chains that participate in Ca
occlusion are indicated by filled circles (Vilsen and
Andersen, 1992d; Andersen and Vilsen,
1994).
In the present study, the roles of the
hydrophobic side chains of Phe, Ile
,
Tyr
, Leu
, and Ile
located at
the border between the fifth transmembrane segment M5 and the putative
``stalk'' helix S5 have been analyzed by substitution of
these residues with glycine. In addition, Leu
was
replaced by lysine to examine the effect of the positive charge on
Ca
binding. These residues are totally conserved
among the SERCA-type of Ca
-ATPases. Other cation
transporting ATPases of P-type contain motifs that are highly similar
although not identical, and the aromatic nature of the residue found at
the place of the tyrosine is well conserved (Green, 1989).
The
results of the functional analysis of the mutants support the notion
that these residues are located close to the Ca binding structure and demonstrate a role of Tyr
in
ensuring the coupling of ATP hydrolysis to Ca
transport. Hence, the Tyr
Gly mutant
catalyzed ATP hydrolysis at a high rate in the absence of any net
accumulation of Ca
in the vesicles.
The construction and expression of mutant cDNAs as well as
most of the methods employed in the analysis of enzyme function have
previously been described in detail (Maruyama and MacLennan, 1988;
Andersen et al., 1989; Andersen and Vilsen, 1992b; Vilsen et al., 1989, 1991; Vilsen and Andersen, 1992b). In brief,
mutations were introduced into the rabbit fast twitch muscle
Ca-ATPase cDNA using the site-specific mutagenesis
method of Kunkel(1985). The presence of the correct mutation was
confirmed by nucleotide sequencing according to Sanger et
al.(1977). The entire Ca
-ATPase cDNA containing
the desired mutation was cloned into vector pMT2 (Kaufman et
al., 1989) for expression in COS-1 cells (Gluzman, 1981).
Microsomes were prepared from transfected cells, and the expression of
exogenous Ca
-ATPase protein was quantitated by a
specific sandwich enzyme-linked immunosorbent assay (Andersen et
al., 1989).
ATP-driven Ca
uptake
in the microsomes was measured by Millipore filtration (Vilsen et
al., 1989) following incubation of 0.05-0.2 µg of
Ca
-ATPase protein at 37 °C for 1-30 min
(depending on the specific transport rate) in medium containing 20
mM MOPS, pH 6.8, 100 mM KCl, 5 mM MgCl
, 5 mM ATP, 0.5 mM EGTA, 5
mM potassium oxalate, 10
Bq
Ca
/ml, and various concentrations of
CaCl
to set the concentrations of free
Ca
at the desired values according to calculations
using published stability constants for Mg
and
Ca
complexes of EGTA, ATP, and oxalate
(Sillén and Martell, 1964; Fabiato and Fabiato,
1979; Dupont, 1982; Vilsen and Andersen, 1987). A
Ca
-selective electrode (Radiometer F2002) was used to
adjust free Ca
concentrations above 10
µM. All solutions were prepared freshly before use and
kept at 37 °C to avoid precipitation of calcium oxalate. Following
subtraction of the background Ca
influx measured with
microsomes harvested from cells transfected with vector without insert,
the rate of Ca
uptake was calculated from the linear
part of the curves showing Ca
uptake as a function of
time (see inset in Fig. 2).
Figure 2:
Ca dependence of the
rate of ATP-driven Ca
uptake in the microsomal
vesicles. Ca
uptake was measured as described under
``Experimental Procedures'' at 37 °C in the presence of
oxalate, and the molecular turnover rate was calculated as the ratio
between the rate of Ca
uptake/mg of microsomal
protein (µmol of Ca
transported/min/mg of
microsomal protein) and the phosphorylation capacity (nmol/mg of
microsomal protein). The ordinate shows this number as
percentage of that measured for the wild-type
Ca
-ATPase at 10 µM Ca
.
The inset shows examples of the time curves from which the
rates were calculated, at pCa 3.8 for the wild-type and mutant
Phe
Gly and pCa 3.7 for mutant Tyr
Gly.
, wild-type Ca
-ATPase;
, mutant Phe
Gly;
, mutant
Ile
Gly;
, mutant Tyr
Gly;
, mutant Leu
Gly;
, mutant
Ile
Gly.
The rate of ATP
hydrolysis was measured spectrophotometrically at 37 °C by the
NADH-coupled assay described by Vilsen et al.(1991), in the
presence of 0.15 mM NADH, 1 mM phosphoenolpyruvate,
lactate dehydrogenase (approximately 10 IU/ml), and pyruvate kinase
(approximately 10 IU/ml). Additional components included in the
standard medium used in most experiments were 20 mM MOPS, pH
7.0, 100 mM KCl, 2 mM MgCl, 5 mM MgATP, 1 mM EGTA, 1 µM of the calcium
ionophore A23187, and various concentrations of CaCl
to set
the desired concentrations of free Ca
. The ATPase
activity referable to the Ca
-ATPase was calculated
following subtraction of the rate of ATP hydrolysis measured in the
absence of free Ca
(presence of 2 mM excess
EGTA). MgATP concentration dependence was titrated as in Vilsen et
al. (1991) keeping the free Mg
concentration
constant at 1 mM and the free Ca
concentration at 100 µM. Inhibition by various
concentrations of vanadate was tested in the presence of 100 µM Ca
and 5 mM MgATP as described in
Vilsen and Andersen (1992b).
The molecular turnover rates were
calculated using the maximum amount of phosphoenzyme formed from ATP
(see below) as a measure of the concentration of active enzyme sites
present. In the figures these rates are presented as percentage of the
maximum molecular turnover rate observed with the wild-type
Ca-ATPase.
The phosphoenzyme intermediates formed
in the presence of [-
P]ATP or
P
were quantitated as a function of
Ca
concentration following acid quenching at steady
state. The phosphorylation from [
-
P]ATP was
carried out at 0 °C for 15 s in the presence of 50 mM MOPS
buffer, pH 7.0, 80 mM K
, 5 mM Mg
, 2 µM [
-
P]ATP, 1 mM EGTA, and
CaCl
to set the indicated free Ca
concentrations. To study the rate of dephosphorylation of the E1P phosphoenzyme intermediate in the absence of
Ca
accumulation in the vesicles, 1 µM of
the calcium ionophore A23187 was added to the standard phosphorylation
medium. Phosphorylation was terminated by the addition of excess EGTA
(1 mM), with or without 1 mM ADP, followed by acid
quenching at serial time intervals. Similar experiments were carried
out following phosphorylation in a medium designed to accumulate the E2P phosphoenzyme intermediate (Andersen et al.,
1989; Vilsen et al., 1989). This medium contained 100 mM TES/Tris buffer, pH 8.35, 10 mM Mg
, 50
µM Ca
, and 2 µM [
-
P]ATP. Phosphorylation from
P
to form E2P in the
``backdoor'' reaction was carried out for 10 min at 25 °C
in the presence of 100 µM
P
, 100
mM TES/Tris, pH 7.0, 5 mM Mg
, 20%
(v/v) dimethyl sulfoxide, and the indicated Ca
concentrations set with EGTA. Dimethyl sulfoxide was added to
increase the apparent affinity of the Ca
-ATPase for
P
(De Meis et al., 1980) so that the
phosphorylation level was close to maximum in the absence of
Ca
. To study the rate of dephosphorylation of E2P formed from
P
, the phosphorylated
sample was cooled to 0 °C. The phosphorylation was then terminated
by a 20-fold dilution of the sample into ice-cold medium containing 50
mM MOPS, pH 7.0, 80 mM K
, 5 mM Mg
, 1 mM non-radioactive P
,
2 mM EGTA, no dimethyl sulfoxide, followed by acid quenching
at serial times.
The phosphorylated acid-precipitated microsomal protein was washed and subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0, followed by autoradiography of the dried gels and quantitation by densitometric analysis, using an LKB 2202 Ultroscan Laser Densitometer, or by liquid scintillation counting of gel slices. The latter procedure was used to obtain the absolute site concentration needed for calculation of molecular turnover rate.
All experiments were repeated at least three times with consistent results. The data points shown in Fig. 2, Fig. 4, and Fig. 5are mean values of triplicate determinations. S.D. is indicated when larger than 5%.
Figure 4:
Ca dependence of ATPase
activity. The ATPase activity was measured in the presence of 1
µM calcium ionophore as described under
``Experimental Procedures'' and illustrated in Fig. 3.
The molecular turnover rate was calculated as the ratio between the
rate of ATP hydrolysis/mg of microsomal protein and the phosphorylation
capacity/mg of microsomal protein. The ordinate shows this
number as percentage of that measured for the wild-type
Ca
-ATPase at 50 µM Ca
and 5 mM MgATP. The concentration of free Ca
vas varied at a constant MgATP concentration of 5 mM.
, wild-type Ca
-ATPase;
, mutant
Phe
Gly;
, mutant Tyr
Gly;
, mutant Leu
Gly;
, mutant
Ile
Gly.
Figure 5:
Ca concentration
dependence of phosphorylation from [
-
P]ATP
or
P
. Phosphorylation of the microsomal
fractions isolated from cells transfected with mutant or wild-type
Ca
-ATPase cDNA was carried out as described under
``Experimental Procedures.'' The acid-quenched samples
containing equivalent amounts of expressed Ca
-ATPase
were subjected to SDS-polyacrylamide gel electrophoresis at pH 6.0. The
autoradiograms of the dried gels were quantitated by densitometry. A, phosphorylation from ATP. B, phosphorylation from
P
.
, wild-type Ca
-ATPase;
, mutant Phe
Gly;
, mutant
Tyr
Gly;
, mutant Leu
Gly;
, mutant Ile
Gly;
, mutant
Leu
Lys.
Figure 3:
Ionophore sensitivity of
Ca-activated ATPase activity. ATP hydrolysis was
monitored spectrophotometrically by the NADH-coupled assay described
under ``Experimental Procedures'' in the presence of 20
mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl
, 5 mM MgATP, and 100 µM Ca
. The ordinate shows absorbance at
340 nm. The bar in the lower left corner indicates 0.1
absorbance unit. At 1, the microsomes were added. At 2, 1 µM of the calcium ionophore was added to
relieve back-inhibition by accumulated Ca
. At 3, EGTA was added to show the background ATPase activity not
referable to Ca
-ATPase. A, wild-type
Ca
-ATPase; B, mutant Tyr
Gly; C, mutant Phe
Gly; D, control microsomes harvested from cells transfected with
vector without insert (note that more microsomal protein was inserted
in this case).
To be able to measure
the rate of ATP-driven Ca transport into the
microsomal vesicles containing expressed Ca
-ATPase,
in the presence of the background of passive diffusion of
Ca
into all vesicles in the preparation, oxalate is
usually added to the reaction mixture. The precipitation of
Ca
oxalate at the high Ca
concentrations generated inside the vesicles during active
transport traps the calcium ions, thereby increasing the capacity for
active Ca
accumulation. Fig. 2shows the
results of measurement of the specific Ca
transport
turnover rates of mutants Phe
Gly, Ile
Gly, Tyr
Gly, Leu
Gly, and Ile
Gly, in the presence of
oxalate. In mutant Ile
Gly, the maximal transport
rate measured at saturating Ca
concentration and the
apparent Ca
affinity were rather similar to those of
the wild-type Ca
-ATPase. On the other hand, the
maximal transport rates of mutants Leu
Gly and
Ile
Gly were reduced below 15% that of the
wild-type precluding an accurate determination of the Ca
affinities of these mutants. Mutant Phe
Gly
was clearly able to transport Ca
at a higher rate,
but since this mutant displayed very low apparent Ca
affinity (K
above 50 µM, see Fig. 2), it was not straightforward to determine its maximum
Ca
-uptake activity. Hence, at high medium
Ca
concentrations Ca
oxalate
crystals are formed not only inside the vesicles, but also outside,
giving rise to a high filter background. For this reason, we have
previously been unable to measure the oxalate-supported Ca
uptake with accuracy at free Ca
concentrations
higher than 10-30 µM, but in the present study it
was found that the high filter background can be avoided up to a free
Ca
concentration of about 200 µM (determined using the Ca
selective electrode),
provided the supersaturated Ca
-oxalate solutions are
prepared immediately before use and the Ca
uptake
measured at 37 °C instead of at 27 °C as previously. As seen in Fig. 2, these precautions permitted the measurement of
ATP-driven Ca
transport in mutant Phe
Gly at Ca
concentrations up to 200
µM Ca
. At this Ca
concentration, the turnover rate corresponded to about 50% that
of the maximum turnover rate of the wild-type.
Fig. 2also
shows that mutant Tyr
Gly was unable to transport
Ca
at a measurable rate at all Ca
concentrations up to the experimental limit of 200
µM. Even after 30 min of incubation at 200 µM Ca
in the presence of 5 mM MgATP, it
was not possible to measure any Ca
uptake higher than
the background corresponding to control microsomal preparations
harvested from cells that had been mock-transfected with the vector
without insert. In addition, the mutant with positively charged
substituent, Leu
Lys, was unable to transport
Ca
at Ca
concentrations up to 200
µM (not shown).
The ATPase
activity was further characterized by measurement of the dependence on
MgATP concentration at saturating Ca concentration
(not shown). The activation curve for the Tyr
Gly
mutant was almost superposable on that of the wild-type, and like the
wild-type Ca
-ATPase, all the mutants displayed a
biphasic activation pattern with a secondary rise in the millimolar
concentration range indicative of a role for ATP as an allosteric
modulator in addition to its role as substrate (Andersen, 1989). In
addition, it was found that all mutants, including Tyr
Gly, were sensitive to inhibition by vanadate.
Studies of the inhibition by Ca of the backdoor phosphorylation from P
may provide
additional information on the function of the Ca
sites (Andersen and Vilsen, 1992b, 1994). The results of such
experiments are depicted in Fig. 5B. A comparison with Fig. 5A indicates that the apparent Ca
affinities determined in the two types of phosphorylation assay
were fairly consistent, except in the case of mutant Phe
Gly. The apparent Ca
affinity observed
by titration of Ca
inhibition of P
phosphorylation in this mutant was almost identical to that of
the wild-type, in contrast to the low Ca
affinity
observed in the study of Ca
activation of
phosphorylation from ATP. Because the Ca
-ATPase is
thought to bind two calcium ions in a consecutive mechanism, and only
the first binding site has to be occupied to inhibit phosphorylation
from P
, a possible explanation of the discrepancy between
the apparent Ca
affinities determined in the two
types of phosphorylation assay with mutant Phe
Gly
is that Phe
is directly or indirectly involved in the
binding of the second calcium ion, but not in the binding of the first
calcium ion in the sequence (Andersen and Vilsen, 1992b, 1994).
Figure 6:
Dephosphorylation of the phosphoenzyme
intermediates formed from [-
P]ATP or
P
. The acid-quenched
P-phosphorylated samples were subjected to
SDS-polyacrylamide gel electrophoresis at pH 6.0, and the
autoradiograms of the dried gels are shown. A, phosphorylation
with [
-
P]ATP was carried out at 0 °C in
the presence of 50 mM MOPS buffer, pH 7.0, 80 mM K
, 5 mM Mg
, 0.1 mM Ca
, 1 µM of the Ca
ionophore A23187, and 2 µM [
-
P] ATP. Following 15-s reaction with
[
-
P]ATP, the sample was acid-quenched
directly (lane 1), or incubated with 1 mM ADP and 1
mM EGTA for 5 s (lane 2), or with 1 mM EGTA
without ADP for 5 s (lane 3) or 10 s (lane 4), prior
to acid-quenching. B, phosphorylation with
P
was carried out at 25 °C for 10 min in
the presence of 100 µM
P
, 100
mM TES/Tris pH 7.0, 5 mM Mg
, 2
mM EGTA, and 20% (v/v) dimethyl sulfoxide. Following cooling
to 0 °C, the sample was acid-quenched directly (0), or
dephosphorylation was initiated by 20-fold dilution of an aliquot into
ice-cold medium containing 50 mM MOPS, pH 7.0, 80 mM K
, 5 mM Mg
, 1 mM non-radioactive P
, and 2 mM EGTA, and acid
quenching was performed 5 or 10 s after the dilution. C,
phosphorylation was carried out at 0 °C for 15 s in the presence of
100 mM TES/Tris, pH 8.35, 10 mM Mg
,
50 µM Ca
, and 2 µM [
-
P]ATP. The phosphorylated sample was
either acid-quenched directly (lane 1), or incubated with 1
mM ADP and 1 mM EGTA for 5 s (lane 2), prior
to acid-quenching. In lane 3, the phosphorylated sample was
incubated with 1 mM EGTA without ADP for 20 s, prior to
acid-quenching. In lane 4, the phosphorylated sample was
incubated with 1 mM EGTA without ADP for 15 s followed by 1
mM ADP for 5 s, prior to
acid-quenching.
Because the E2P
phosphoenzyme intermediate is likely to represent the enzyme form that
releases Ca at the luminal surface, a plausible
explanation for the uncoupling of ATP hydrolysis from Ca
transport in mutant Tyr
Gly would be that E1P is hydrolyzed directly to produce P
, thereby
bypassing E2P. To test the ability of mutant Tyr
Gly to form E2P in the forward direction from E1P, phosphorylation from ATP was in addition carried out at
alkaline pH and in the absence of alkali metal ions. Under these
conditions E2P accumulates as the major steady state
intermediate in the wild-type Ca
-ATPase, due to a
reduced rate of dephosphorylation of E2P (Andersen et
al., 1989; Vilsen et al., 1989). It is seen in Fig. 6C, lanes 1 and 2, that the E2P phosphoenzyme intermediate was accumulated in the
Tyr
Gly and Leu
Gly mutants
as well, as demonstrated by the fraction of phosphoenzyme being
insensitive to ADP. Lanes 3 and 4 demonstrate that
the ADP-insensitive fraction of the phosphoenzyme increased further in
the Tyr
Gly mutant during a 15-s incubation with
EGTA.
The present study features mutant Tyr
Gly as a new phenotypic variant of the Ca
-ATPase
which is uncoupled in the sense that it hydrolyzes ATP at a high rate
in a Ca
-activated reaction without any net
accumulation of Ca
in the microsomal vesicles. In
this mutant, phosphorylation from ATP and the rate of hydrolysis of ATP
were maximally stimulated by Ca
at a Ca
concentration as low as 10 µM, whereas no
Ca
accumulation was detected at Ca
concentrations up to the experimental limit of 200
µM. The uncoupling of ATPase activity from Ca
accumulation in the Tyr
Gly mutant was
furthermore documented by the lack of an activatory effect of ionophore
on the ATP hydrolysis.
The uncoupled ATP hydrolysis catalyzed by the
Tyr
Gly mutant proceeded through formation of an
acid-stable phosphoenzyme intermediate and displayed characteristics
very similar to the ionophore-activated ATPase activity of the
wild-type Ca
-ATPase with respect to specific rate,
MgATP concentration dependence, and vanadate inhibition. In the
wild-type enzyme, dephosphorylation takes place through the partial
reaction sequence E1P
E2P
E2
+ P
. It was directly demonstrated that the Tyr
Gly mutant is able to form the E2P intermediate
from E1P (Fig. 6C). Because E2P is
the intermediate thought to release Ca
at the luminal
surface (De Meis, 1981; Andersen, 1989; McIntosh et al.,
1991), it is possible that the machinery responsible for the
translocation of Ca
is intact in the Tyr
Gly mutant, and that the lack of net accumulation of
Ca
by the mutant is due to an increased rate of
efflux of transported Ca
from the vesicles. Such an
increased passive efflux of Ca
would be reminiscent
of the leak of Ca
through the
Ca
-ATPase observed following binding of
phenothiazines and local anesthetics to the pump (De Meis, 1991;
Wolosker et al., 1992; De Meis and Inesi, 1992). It was
suggested that this efflux could be mediated through stabilization of
unphosphorylated reaction intermediates in the late part of the normal
reaction cycle (De Meis, 1991).
Alternatively, the calcium ions are
not translocated by the Tyr
Gly mutant due to a
defect in the early part of the reaction cycle. It is possible that the
gate normally preventing the occluded calcium ions from dissociating to
the cytoplasmic side upon phosphorylation is defective in the mutant so
that an anomalous E1P intermediate with non-occluded
Ca
sites is created. The side chain of Tyr
might play a critical role in the gating mechanism, not unlike
the roles of the aromatic side chains in pore regions of voltage-gated
ion channels (Heginbotham and MacKinnon, 1992; Miller, 1993). It is
also possible that the removal of the bulky side chain of the tyrosine
led to a less specific and more widespread disturbance of the
transmembrane domain, for instance as a consequence of changes in helix
packing.
The slight reduction of apparent Ca affinity of the uncoupled Tyr
Gly mutant as
well as the more pronounced changes in Ca
affinity
observed with mutants Phe
Gly and Leu
Lys are in accordance with the hypothesis (Fig. 1)
that the M5S5 boundary is located in the vicinity of the entrance to
the Ca
-binding domain. The effect of the positively
charged lysine side chain on Ca
binding can be
explained by electrostatic repulsion of the calcium ions.
The mutant
Leu
Lys was able to dephosphorylate at a normal
rate, while the dephosphorylation of the E2P intermediate was
blocked in mutants Leu
Gly and Ile
Gly, and more so in the latter mutant. The block of E2P dephosphorylation explains the low ATPase activity
observed with mutants Leu
Gly and Ile
Gly. A similar block of dephosphorylation of E2P
has been observed for other mutants with alterations to residues in
transmembrane segments M4, M5, and M6, including the putative
Ca
liganding residues Glu
,
Glu
, and Asn
(Andersen and Vilsen, 1992b,
1994; Vilsen and Andersen, 1992c; Clarke et al., 1993). Since
recent evidence suggests that protons are countertransported by the
Ca
-ATPase (Levy et al., 1990; Yu et
al., 1993), very likely in a way similar to the K
transport by Na
,K
-ATPase, we
suggested that the role for these residues in the dephosphorylation of E2P might be associated with the donation of the side chain
oxygen to form binding/occlusion site(s) for the H
(possibly H
O
) to be
countertransported (Andersen and Vilsen, 1992b). It is possible that
the bulky side chains of Leu
and Ile
are
crucial to the dephosphorylation of E2P because they stabilize
the proton binding structure or because they participate in the
conformational change(s) mediating long range communication with the
phosphorylation site. The recovery of the ability to dephosphorylate
rapidly upon exchange of the glycine substituent with lysine might be
ascribed solely to the bulk of the side chain of the lysine. It is,
however, also possible that the presence of a positively charged side
chain near the suggested proton-binding site mimicks a proton, thereby
inducing dephosphorylation.
Finally, the ATPase activity of mutant
Leu
Gly was higher than that of mutant Ile
Gly, while the corresponding Ca
uptake
was lower, indicating that also mutant Leu
Gly may
have been uncoupled to some extent.
In summary, the present study
has pointed to the hydrophobic residues at the M5S5 boundary as
important for the tight coupling between ATP hydrolysis and
Ca transport, and for the partial reactions involved
in Ca
binding and E2P dephosphorylation. It
will be important to try in the future to further resolve the reasons
for the observed uncoupling.