(Received for publication, June 26, 1995; and in revised form, August 8, 1995)
From the
We have determined the stoichiometry and rate constants for the
dissociation of Ca ion from calmodulin (CaM)
complexed with rabbit skeletal muscle myosin light chain kinase
(skMLCK), rat brain nitric oxide synthase (nNOS) or with the respective
peptides (skPEP and nPEP) representing the CaM-binding domains in these
enzymes. Ca
dissociation kinetics determined by
stopped-flow fluorescence using the Ca
chelator
quin-2 MF are as follows. 1) Two sites in the CaM-nNOS and CaM-nPEP
complexes have a rate constant of 1 s
. 2) The
remaining two sites have a rate constant of 18 s
for
CaM-nPEP and >1000 s
for CaM-nNOS. 3) Three sites
have a rate constant of 1.6 s
for CaM-skMLCK and
0.15 s
for CaM-skPEP. 4) The remaining site has a
rate constant of 2 s
for CaM-skPEP and >1000
s
for CaM-skMLCK. Comparison of these rate constants
with those determined for complexes between the peptides and tryptic
fragments representing the C- or N-terminal lobes of CaM indicate a
mechanism for Ca
dissociation from CaM-nNOS of 2C
slow + 2N fast and from CaM-skMLCK of (2C + 1N) slow +
1N fast. Ca
removal inactivates CaM-nNOS and
CaM-skMLCK activities with respective rate constants of >10
s
and 1 s
. CaM-nNOS inactivation
is fit by a model in which rapid Ca
dissociation from
the N-terminal lobe of CaM is coupled to enzyme inactivation and slower
Ca
dissociation from the C-terminal lobe is coupled
to dissociation of the CaM-nNOS complex. CaM-skMLCK inactivation is fit
by a model in which the three slowly dissociating
Ca
-binding sites are coupled to both dissociation of
the complex and enzyme inactivation.
An understanding of cellular regulation will require elucidation
of the different kinetic mechanisms by which CaM ()regulates
the many enzyme activities under its control. In this respect it is
clear that different structural domains in CaM contribute quite
variably to both the activation and binding of different target enzymes
(George et al., 1990; Persechini et al., 1994). For
example, it was recently demonstrated that fragments representing the
N- and C-terminal lobes of CaM, each of which contains one of the two
pairs of EF-hand Ca
-binding sites in CaM, bind to
their respective sites on neural nitric oxide synthase (nNOS) and
skeletal muscle myosin light chain kinase (skMLCK) with affinities
differing by a factor of 100 or more between the two enzymes, although
the concentrations of intact CaM required for half-maximal activation
of these enzyme activities are not significantly different (Persechini et al., 1994).
Olwin et al.(1984) and Olwin and
Storm(1985) have demonstrated free energy coupling between target and
Ca binding to CaM such that the
Ca
-binding affinity of CaM in its complex with the
target is increased over what is seen with free CaM in proportion to
the relative increase in target binding affinity caused by
Ca
binding to CaM. Our recent results with CaM
tryptic fragments suggest that the free energy coupling associated with
Ca
-dependent binding of CaM to nNOS and skMLCK is not
equally distributed among the four Ca
-binding sites
of CaM. As a result, the roles of the two CaM lobes in establishing
Ca
-dissociation and enzyme inactivation rates for the
CaM-nNOS and CaM-skMLCK complexes should differ significantly.
In
this study we have investigated the rates of Ca dissociation from the CaM-nNOS and CaM-skMLCK complexes and the
rates of enzyme inactivation associated with this process. We have
confirmed substantial differences between the mechanisms of
Ca
dissociation from the two CaM-enzyme complexes
that are attributable to the distinct roles played by the CaM lobes in
these complexes.
Vertebrate CaM was expressed in Escherichia coli and purified as described by Persechini et al.(1989). skMLCK was expressed in Sf9 cells and purified as described by Fitzsimmons et al.(1992). nNOS was expressed in HEK A293 cells and purified essentially as described by McMillan et al.(1992). CaM tryptic fragments TRCI and TRCII were generated and purified as described by Persechini et al.(1994). Synthetic peptides; KRRAIGFKLAEVKFSAKLMGQ-amide (nPEP), based on the reported CaM-binding domain in nNOS (Vorher et al., 1993; Zhang and Vogel, 1994), and KRRWKKNFIAVSAANRFKK-amide (skPEP), based on the reported CaM-binding domain in skMLCK (Blumenthal and Krebs, 1987), were commercially synthesized (Quality Controlled Biochemicals, Inc) and were verified by mass spectrometry.
Enzyme inactivation experiments
were performed at 25 °C with a pneumatically driven three syringe
mixing device built and used as described by McCarthy et
al.(1994). Reactions containing 1 µM CaM and 650
nM nNOS or 50 nM skMLCK were initiated by adding L-[2,3,4,5-H]arginine (50,000 cpm/pmol;
Amersham Corp.) or [
-
P]ATP (1000 cpm/pmol;
DuPont NEN) to final concentrations of 1.4 µM or 1
mM, respectively. The reaction buffer for both enzyme
reactions contained 50 mM MOPS, 150 mM NaCl, 1 mM dithiothreitol, 200 µM CaCl
, and 10
mM MgCl
, pH 7.0. For nNOS reactions the buffer
also contained 1 µM tetrahydrobiopterin and 100 µM NADPH. Reactions were quenched at the indicated times by addition
of 1 volume of either 150 mM phosphoric acid (skMLCK) or 15%
trichloroacteic acid (nNOS). Aliquots of the quenched samples were
processed to quantitate amounts of product as described previously
(Persechini et al., 1994). For measurements of enzyme
inactivation rates, samples were quenched at various times after
addition of EGTA to a final concentration of 3 mM. Theoretical
curves for enzyme inactivation were generated using an integrated rate
equation where P is the amount of product, V is the
linear enzyme rate prior to EGTA addition, k
is the first-order rate of inactivation after EGTA addition, t is the time after addition of the chelator, and C is the amount of product present when EGTA is added to the
reaction mixture.
Curve fitting to data for enzyme inactivation time courses was performed using the Prism software package (GraphPad, Inc.)
Presteady-state measurements of changes in quin-2 MF (Molecular Probes) fluorescence observed at 90° to the excitation beam were performed using a stopped-flow fluorimeter with a dead time of 1.5 ms (Applied Photophysics, Leatherhead, United Kingdom). The quin-2 MF excitation wavelength at 320 nm was obtained using a 0.124-m monochrometer (Farrand Corp.) illuminated by a 100-watt mercury arc lamp. Fluorescence emission from quin-2 MF was isolated with a 520 nm high-pass glass filter (Corion). Four or more stopped-flow time courses of 1024 data points were averaged for each experiment. Data were filtered with a time constant of 0.4 ms and collected with a Nicolet Explorer III digital oscilloscope and finally transferred to a Zenith HL-148 PC for permanent storage and analysis. Observed rate constants were obtained by fitting the data to single or double exponential equations by the method of moments (Dyson and Isenberg, 1971) or using the Prism software package (GraphPad, Inc.). Stopped-flow experiments were performed at 25 °C.
For measurements of Ca dissociation from CaM-nNOS or CaM-skMLCK complexes, syringe A of
the stopped-flow apparatus contained 2 µM CaM, a molar
excess of enzyme over CaM, and 50 µM CaCl
.
Enzyme was omitted for measurements of Ca
dissociation from CaM alone. Syringe B contained 200 µM quin-2 MF. Both syringes contained 150 mM NaCl, 20 mM MOPS, 0.5 mM dithiothreitol, pH 7.0. Similar conditions
were used for measurements of Ca
dissociation from
complexes between nPEP or skPEP and CaM, TRCI, or TRCII except that the
concentration of CaM or CaM fragment in syringe A was 6 or 5
µM, respectively. Measurements were made at 2 or more
peptide:CaM stoichiometries. Measurements of the decay in intrinsic Trp
fluorescence of the CaM-skPEP complex were made under conditions
identical to those described above, except the excitation wavelength
was 295 nm and fluorescence emission from Trp was isolated with a
320-380 nm band-pass glass filter (Oriel Corp.), and
Ca
was chelated with EDTA at a final concentration of
1 mM instead of with quin-2 MF. In all cases reactions were
initiated by mixing equal volumes from syringe A and B so that the
final concentrations of protein, CaCl
, and quin-2 MF were
half of their initial values.
Calibration of the photomultiplier
voltage with respect to increases in Ca was performed
by mixing a set of CaCl
standards with buffer containing
the quin-2 MF indicator. This indicator was chosen in preference to
quin-2 because of its 5-fold higher affinity for Ca
ion (K
= 25 nM;
Haughland (1992)). The dependence of the photomultiplier voltage upon
added free Ca
was found to be linear with a slope of
57 mV/µM added CaCl
. This slope was used to
determine the stoichiometry of Ca
release from CaM,
TRCII, and TRCI. A reference for Ca
-free indicator
obtained by mixing the indicator with 10 mM EDTA provides an
estimate of free Ca
in the buffer of 2
µM. Quin-2 has been reported to bind Ca
ion with an association rate constant of 7.5
10
M
s
, so
at the final concentration of 100 µM used in stopped-flow
experiments the rate of Ca
binding to quin-2 would
exceed any measurable Ca
dissociation rate by at
least a factor of 100 (Bayley et al., 1984).
150 mM NaCl was added to experimental buffers to prevent
Ca-independent interactions between CaM and peptides,
which have been reported to occur at lower ionic strengths (Itakura and
Iio, 1992). We have found that interactions betwen CaM and a peptide
based on the CaM-binding domain in skMLCK are not completely reversed
by Ca
chelation unless 150 mM NaCl is
present. (
)Salt at this concentration does not significantly
affect the ability of CaM to activate nNOS or skMLCK activities (data
not shown). The rate constant for EGTA-induced inactivation of skMLCK
activity we report here (1.0 s
) is not significantly
different from the value of 0.8 s
determined by
Stull et al.(1985) in the absence of added NaCl.
Data for P incorporation into peptide substrate
by skMLCK are presented in Fig. 1. The time course for this
reaction is linear over the experimental interval of 5 s (Fig. 1, inset). Chelation of Ca
ion
by addition of EGTA 3 s after initiation of the phosphorylation
reaction causes a relatively slow inactivation of skMLCK activity (Fig. 1, inset). Fitting data collected during the
first 2 s of this inactivation phase gives an inactivation rate
constant of 1 s
(Fig. 1). As seen with skMLCK
activity, nNOS activity is linear over the experimental time interval;
however, EGTA addition causes a more rapid inactivation that is too
fast to measure. As seen in Fig. 2, an inactivation rate
constant of 1 s
gives a poor fit to the data. A rate
constant of 10 s
also appears too slow to describe
the inactivation process, while a rate constant of 25 s
or greater appears to provide a reasonable fit. Based on this
analysis, we conservatively estimate that the rate constant for
inactivation of nNOS activity by addition of EGTA is >10
s
.
Figure 1:
Inactivation of skMLCK activity by EGTA
addition. Data are shown for P incorporation into peptide
substrate catalyzed by skMLCK after addition of 3 mM EGTA at t = 3 s. Concentrations of skMLCK and CaM were 50
nM and 1 µM, respectively. The initial
Ca
concentration was 200 µM. The curve
shown in the figure was calculated from using a k
value of 1 s
. Inset, data are shown for a 5-s time course of
P
incorporation catalyzed by skMLCK with (
) or without (
)
addition of 3 mM EGTA at the indicated time. Error bars represent the standard error of the mean (n =
3).
Figure 2:
Inactivation of nNOS activity by EGTA
addition. Data are shown for L-[H]citrulline production catalyzed by
nNOS with (
) or without (
) addition of 3 mM EGTA
at the indicated time. Concentrations of nNOS and CaM were 650 nM and 1 µM, respectively. The initial Ca
concentration was 200 µM. Theoretical curves for
enzyme inactivation were calculated according to using
rate constants of 1 s
(A), 10
s
(B), and 25 s
(C). Error bars represent the standard error of the mean (n = 3).
Data for dissociation of Ca ion from the CaM-skMLCK and CaM-nNOS complexes are presented in Fig. 3A. The data are fit well by single exponentials
with respective rate constants of 1.6 and 1 s
.
However, the amplitudes of the signals for the two CaM-enzyme complexes
are clearly different. For the CaM-skMLCK complex, an amplitude of 2.8
mol of Ca
/mol of CaM is obtained; the corresponding
amplitude for the CaM-nNOS complex is 1.9 mol of
Ca
/mol of CaM (Fig. 3A). Data for
Ca
release from free CaM are fit by a single
exponential with a rate constant of 12.6 s
and an
amplitude of 1.9 mol of Ca
/mol of CaM.
Figure 3:
Release of Ca ion from
CaM-enzyme or CaM-peptide complexes. Quin-2 MF fluorescence data were
converted to Ca
release stoichiometries using a ratio
of 57 mV/µM Ca
determined as described
under ``Materials and Methods.'' The final quin-2 MF
concentration was 100 µM. Panel A contains data
for Ca
release from CaM-skMLCK and CaM-nNOS after
addition of quin-2 MF. The final CaM concentration was 1
µM, with final skMLCK and nNOS concentrations of 1.3
µM and 1.9 µM, respectively. Panel B contains data for Ca
release from CaM-nPEP and
CaM-skPEP after addition of quin-2 MF. The final CaM concentration was
3 µM with final skPEP and nPEP concentrations of 5
µM. Curves are single or double exponentials calculated
using the rate constants and amplitudes given in Table 1.
In contrast
with free CaM or the CaM-enzyme complexes, we were able to observe
Ca dissociation from all four
Ca
-binding sites in the CaM-peptide complexes. Data
for Ca
release from the CaM-nPEP and CaM-skPEP
complexes are presented in Fig. 3B. Data for only the
first 3 s are shown so that the fast phase of Ca
release from CaM-nPEP can be seen. Data for Ca
release from CaM-nPEP are fit by a double exponential with rate
constants of 1 and 17.7 s
, and amplitudes of 1.8 and
2.1 mol of Ca
/mol of CaM, respectively (Fig. 3B). Data for CaM-skPEP are fit by a double
exponential with rate constants of 0.15 and 1.9 s
and amplitudes of 2.8 and 0.9 mol of Ca
/mol of
CaM, respectively (Fig. 3B).
In an attempt to match
observed Ca dissociation rates with one or the other
of the two CaM lobes, we investigated the kinetics of Ca
release from TRCI or TRCII either alone or in the presence of a
molar excess of nPEP or skPEP. Data for dissociation of Ca
ion from free TRCII are fit by a single exponential with a rate
constant of 16 s
and an amplitude of 2 mol of
Ca
/mol of TRCII; Ca
release from
free TRCI is too fast to measure. In the presence of a molar excess of
skPEP, Ca
release from TRCII is described by a single
exponential with a rate constant of 0.34 s
and an
amplitude of 2 mol of Ca
/mol of TRCII. Under these
conditions, release of Ca
ion from the TRCI is
described by a single exponential with a rate constant of 4.3
s
and an amplitude 2.1 mol of
Ca
/mol of TRCI (Fig. 4). In the presence of a
molar excess of nPEP, Ca
release from TRCII is
described by a single exponential with a rate constant of 1.1
s
and an amplitude of 2.2 mol of
Ca
/mol of TRCII. Release of Ca
ion
from the TRCI under these conditions is described by a single
exponential with a rate constant of 100 s
and an
amplitude of 1.8 mol of Ca
/mol of TRCI (Fig. 4).
Figure 4:
Release of Ca ion from
TRCI- or TRCII-peptide complexes. Quin-2 MF fluorescence data were
converted to Ca
release stoichiometries as in Fig. 3. The final quin-2 MF concentration was 100
µM. The final concentration of either TRCI or TRCII was
2.5 µM with final peptide concentrations of 5 µM (skPEP) or 10 µM (nPEP). Curves are single
exponentials calculated using the rate constants and amplitudes given
in Table 1. Panel A contains data for TRCI-skPEP,
TRCII-nPEP, and TRCII-skPEP. Because of their much smaller time scale,
data for TRCI-nPEP are shown separately in panel
B.
It is well established that on binding CaM the
single Trp residue in skPEP undergoes a fluorescence enhancement and
its emission wavelength is blue-shifted due to its interaction with the
C-terminal lobe in CaM (Blumenthal and Krebs, 1987; Ikura et
al., 1992). We therefore examined the rate of decrease in Trp
fluorescence seen during Ca release from the
CaM-skPEP complex. We found this process to be described by a single
exponential with a rate constant of 0.22 s
(data not
shown).
We have found that, when CaM is complexed with skMLCK, the
apparent Ca dissociation rate constant for two sites
in CaM is reduced from 12.6 to 1.6 s
and the rate
constant for 1 site is reduced from > 1000 s
to
1.6 s
. With the CaM-nNOS complex, there is a similar
reduction in the Ca
dissociation rate of the two more
slowly dissociating sites (Table 1). Our results indicate that
the kinetic mechanisms for Ca
release from the
CaM-enzyme complexes are established by interactions present in both
the CaM-enzyme and CaM-peptide complexes. Hence, the CaM-peptide
complexes provide models for elucidating the structural basis for the
distinct mechanisms of Ca
release followed by
CaM-nNOS and CaM-skMLCK.
Kasturi et al.(1993) have
determined the rate of Ca dissociation from the
complex between wheat germ CaM and avian gizzard myosin light chain
kinase using a quin-2 indicator. These investigators have reported that
the rate of Ca
release from free wheat germ CaM is
decreased 5-fold when it is bound to the gizzard kinase; a similar
decrease was reported to occur when CaM is complexed with mellitin
(Kasturi et al., 1993). Although no attempt to determine
Ca
release stoichiometries was made, it was reported
that the amplitude for the observed quin-2 fluorescence transient is
increased 2-fold when CaM is complexed with gizzard myosin light chain
kinase (Kasturi et al., 1993). These observations suggest that
the properties of the CaM-gizzard myosin light chain kinase complex
with respect to Ca
release and inactivation are
likely to be similar to what we report here for the CaM-skMLCK complex.
In order to locate the observed Ca-binding sites
in CaM-nNOS or CaM-skMLCK in one or the other of the two CaM lobes, we
determined Ca
dissociation rates for the complexes
between TRCI or TRCII and either of skPEP or nPEP. We used the
CaM-peptide complexes for these determinations because they follow
kinetic patterns similar to those seen with the CaM-enzyme complexes (3
slow + 1 fast for CaM-skMLCK and 2 slow + 2 fast for
CaM-nNOS), but slower dissociation rates allow all four
Ca
-binding sites in CaM to be observed. Dissociation
of 2 Ca
ions from the TRCII-nPEP complex occurs with
an apparent rate constant essentially identical to the slower
Ca
dissociation rate constant for the CaM-nPEP
complex (Table 1). Two Ca
ions dissociate from
the TRCI-nPEP complex with an apparent rate 5-fold faster than the
faster rate for the CaM-nPEP complex (Table 1). These
observations indicate that the more slowly dissociating pair of sites
in the CaM-nPEP and CaM-nNOS complex is located in the C-terminal CaM
lobe. Analysis of the CaM-skPEP complex is complicated by the
observation that 3 Ca
ions dissociate with a single
apparent rate: 2 from one CaM lobe and 1 from another (Table 1).
Ca
dissociation from the TRCII-skPEP complex occurs
with a rate 2-fold faster than the slower rate for the CaM-skPEP
complex and 5-fold slower than the faster dissociation rate for this
complex (Table 1). Ca
dissociates from the
TRCI-skPEP complex twice as fast as the faster rate for the CaM-skPEP
complex and 10-fold faster than the slower dissociation rate for this
complex (Table 1). This suggests that two of the three more
slowly dissociating Ca
-binding sites in the CaM-skPEP
or CaM-skMLCK complexes are located in the C-terminal lobe of CaM.
Similarly to what we report here, Suko et al.(1986) and
Itakura and Iio(1992) have reported that CaM binding to mellitin
reduces the Ca
dissociation rates for all four sites
in CaM, with the more slowly dissociating sites localized in the
C-terminal lobe of CaM.
Inactivation of the CaM-dependent skMLCK
activity upon removal of Ca ion occurs with a rate
constant of 1 s
; inactivation of CaM-dependent nNOS
activity is too fast to measure (>10 s
).
Dissociation of the CaM-skMLCK complex has been reported to occur with
an apparent rate constant of 2 s
(Johnson et
al., 1981), and kinetic measurements of intrinsic tryptophan
fluorescence suggest that the CaM-skPEP complex dissociates at a rate
of 0.22 s
, close to the slow rate for Ca
release from this complex (Table 1). These observations
suggest that slow release of 3 Ca
ions from the two
CaM lobes in a concerted manner is coupled to both inactivation and
dissociation of the CaM-skMLCK complex. Inactivation of CaM-nNOS
catalytic activity occurs at least 10-fold faster than the rate of
Ca
dissociation from the two sites in the C-terminal
lobe of CaM in this complex. This suggests that Ca
dissociation from the N-terminal lobe of CaM is coupled to
inactivation of nNOS activity, while dissociation of Ca
from the C-terminal lobe is coupled to dissociation of the
CaM-nNOS complex. This is consistent with the apparent dissociation
constant of 10 nM for the
(Ca
)
TRCII-nNOS complex (Persechini et
al., 1994). Given a probable association rate of 10
M
s
(Bowman et
al., 1992; Meyer et al., 1992; Kasturi et al.,
1993), then when saturated with Ca
ion the C-terminal
lobe in CaM would be expected to dissociate from nNOS with a rate
constant of 0.1 s
, 10-fold slower than the rate
constant for Ca
release from this lobe.
This study
demonstrates substantial differences between the CaM-skMLCK and
CaM-nNOS complexes with respect to the kinetic mechanisms for
Ca dissociation and associated inactivation of enzyme
activity. These differences reflect the different contributions of the
two CaM lobes to the stabilities and catalytic activities of these
complexes (Persechini et al., 1994). A surprising aspect to
these results is the observation that 3 Ca
ions
dissociate from the CaM-skMLCK complex in a concerted manner. This may
be a reflection of interactions between the CaM lobes that are evident
in the complex between CaM and a peptide based on the CaM-binding
domain in skMLCK (Ikura et al., 1992). Alternatively, it may
be due to slowing of the dissociation rate for one Ca
from the N-terminal lobe due to specific interactions between
this lobe and the peptide sequence. The differences between the
CaM-skMLCK and CaM-nNOS complexes we report here indicate that these
enzyme activities have distinct spatio-temporal distributions in
response to waves in the intracellular Ca
ion
concentration. This is an important dimension to our understanding of
CaM function, further illuminating how this seemingly simple protein
might shape complex and subtle cellular responses.