The kinetics of nucleotide binding to the
uncoupling protein (UCP) from brown adipose tissue mitochondria were
studied with a filter binding method. Fast and slow phases of binding
were observed, corresponding to the two-stage binding model based on equilibrium binding studies (Huang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Reaction
1).
Although this method determines total binding, only the slow
phase can be resolved. The fast unresolved phase represents the formation of the initial loose UCP-nucleotide complex (UN;
Kd
2 µM), whereas the slow phase
reflects the tight binding (U*N) associated with a conformational
change induced by the bound nucleotide. Best fits of the binding data
yielded, for the slow phase, k+1 values of
3.0 × 10
3 s
1 for GTP, 4.8 × 10
3 s
1 for ATP, 0.13 s
1 for
GDP, and >0.7 s
1 for ADP and dissociation rate constants
(k
1) of 0.10 × 10
3
s
1 for GTP, 0.58 × 10
3
s
1 for ATP, 8.8 × 10
3
s
1 for GDP, and >0.3 s
1 for ADP at pH 6.7 and 4 °C. The rates were fairly pH- and
temperature-dependent. The distribution constant
Kc
(=k+1/k
1) between the
tight and loose complexes ranged between 2 and 30, suggesting formation
of 71-97% of the tight complex at equilibrium. The
Kc
decreases with increasing pH, indicating a
progressively less tight complex population. Anions
(SO42
) form a loose complex with UCP, thus
affecting the initial association step, but not the subsequent
transition step. While the kinetic constants were verified by dilution
and chase experiments as well as in mass action plots, they were
further corroborated with data obtained by fluorescence competition
measurements. Taken together, our results show that nucleotide binding
to UCP occurs via a two-stage mechanism in which the initial loose
complex rearranges slowly into a tight complex.
 |
INTRODUCTION |
Nucleotide binding of the uncoupling protein
(UCP)1 from brown adipose
tissue mitochondria has been key in the isolation and characterization
of this H+-transporting protein (1, 2). By facilitating the
transport of H+ into the mitochondrial matrix, UCP
dissipates the electrochemical energy into heat, a function that has
been related to the non-shivering thermogenesis of this specialized
tissue (3-5). The uncoupling effect is regulated by long-chain fatty
acids as activators and by purine nucleoside di- and triphosphates as
inhibitors (reviewed in Refs. 6 and 7).
Several studies have shown that nucleotide binding to UCP exhibits a
strong pH dependence (5, 8-10). The pH dependence has been attributed
to the interplay of H+-dissociating groups of both the
protein and nucleotide. It was suggested that deprotonation of an
acidic group (Asp/Glu) and of a putative His abolishes electrostatic
interactions between the protein and nucleotide, whereas deprotonation
at the terminal phosphate of the nucleotide enhances its electrostatic
interaction with the protein (9, 10). Kinetic measurements with
fluorescent dansylated nucleotides further show that protonation at
Asp/Glu is a prerequisite for nucleotide binding, whereas nucleoside
triphosphate binding requires formation of an additional positive
charge (HisH+) at the binding cleft (10).
Despite such progress in equilibrium binding studies, a further
understanding of the mechanism of nucleotide binding requires more
insight into the binding kinetics. In this work, we have employed a
filter binding method to evaluate the slow kinetics of nucleotide
binding. With this method, the sum of tight and loose binding was
measured. These data were complemented by kinetic measurements using
specific fluorescent nucleotide probes. The results were analyzed in
terms of a two-stage binding model derived previously (11).
 |
EXPERIMENTAL PROCEDURES |
Materials--
Nucleotides were purchased from Boehringer
Mannheim and NEN Life Science Products. 2
-O-Dansylated
nucleotides were synthesized as described (10). The Millipore
Ultrafree-MC 30,000 NMWL filter unit was obtained from Millipore Corp.
(Bedford, MA). Triton X-100 was from Sigma.
Isolation of UCP--
UCP was isolated from a Triton
X-100-soluble lysate of brown adipose tissue mitochondria according to
the method of Lin and Klingenberg (2). The isolated UCP migrated as a
single Coomassie Blue band at 33 kDa on SDS gel. Protein concentration
was determined according to the method of Lowry et al. (12)
using bovine serum albumin as a standard. Nucleotide-binding sites were
assessed by [14C]GTP binding with an anion-exchange
method (13).
Nucleotide Binding Measured by Filter Binding Method--
UCP
(~300 µg/ml) and radiolabeled nucleotide (1.2-12 µM)
were incubated in an Eppendorf cup for 0.5-180 min in a medium
containing 12 mM Mops, 20 mM
Na2SO4, and 0.1 mM EDTA, pH 6.7, and 4 °C or as otherwise stated. At different time intervals, 100 µl of the mixture was withdrawn, applied to the filter, and
centrifuged immediately for 1 min at 4 °C. 10-20 µl of filtrate
was then taken for scintillation counting.
The binding data showed a biphasic time course of increase. The initial
fast phase could not be resolved by this method, whereas the slow phase
exhibited an exponential increase. In line with our previous results,
we interpret the fast phase as formation of the initial loose
UCP-nucleotide complex (UN) and the slow phase as formation of the
tight complex (U*N). Utilizing our previous two-stage binding model
(11), a simplified reaction scheme is written as shown in Reaction 1 in
the Summary. Kd is an apparent dissociation constant
in the presence of Na2SO4, which was normally
included (8). We assume that the rate-limiting step is the slow
conformational change, and UN is formed so fast that a static
equilibrium is maintained. Since the free nucleotide concentration was
measured, the concentration of bound nucleotide, which is calculated as
the concentration of total nucleotide minus the concentration of free
nucleotide, represents the sum of the concentrations of both loose and
tight complexes (UN + U*N).
The rate of U*N formation is written as shown in Equation 1.
|
(Eq. 1)
|
Thus, Equation 2 follows,
|
(Eq. 2)
|
with Kc
= k+1/k
1.
Applying mass conservation, we have Equations 3 and 4.
|
(Eq. 3)
|
|
(Eq. 4)
|
Thus, N = No/(1 + U/Kd + aU/Kd). Substitution into Equation 4 and
solving for U, we have Equations 5-8.
|
(Eq. 5)
|
|
(Eq. 6)
|
|
(Eq. 7)
|
|
(Eq. 8)
|
For the mass action plot, the concentration of bound nucleotide
is calculated as shown in Equation 9.
|
(Eq. 9)
|
The concentration of free nucleotide (N) is obtained from
Equations 3 and 5. The x axis in the mass action plot
(Bound/free) is thus calculated as shown in Equation 10.
|
(Eq. 10)
|
Least-square fitting was performed with the MicroCal Origin
program (Version 2.75, MicroCal Software Inc.). Computation of the
theoretical curves was achieved with a Turbo Pascal program (Version
6).
Kinetics of Nucleotide Binding Measured by Competition with
Fluorescent Nucleotide Derivatives--
Fluorescence was measured with
a Perkin-Elmer MPF-44A fluorescence spectrophotometer (10). The
excitation and emission wavelengths were 360 and 515 nm, respectively.
The slits were set at 6 nm.
The transition from a loose to a tight complex was slow and could be
conveniently monitored by competition with fluorescent nucleotides. The
transition rate constant (k+1) was measured by
competition with dansyl-ADP in 20 mM Mes, pH 6.8, at
14.5 °C. Thus, a low concentration (2-4 µM) of
nucleotide (ATP, ADP, GTP, or GDP) was added to 0.2 µM
UCP presaturated (>95%) with 14 µM dansyl-ADP. Under
such conditions, the rate of fluorescence decrease was limited by the
rate of nucleotide binding since
k+1·[UCP][No]
k
1dansyl-ADP·[UCP
dansyl-ADP]
k+1dansyl-ADP·[UCP][Do],
where [No] and [Do] are the concentrations of the
competing nucleotide and dansyl-ADP, respectively. The
dissociation was followed by competition with dansyl-GTP. For
this purpose, UCP (0.2 µM) was first saturated (>95%)
with 2-4 µM nucleotide. Then, a large excess of
dansyl-GTP (20 µM) was added to displace the bound
nucleotide. The observed fluorescence increase reflected the
rate-limiting dissociation of the nucleotide from UCP since the kinetic
limitation conditions k+1dansyl-GTP·[dansyl-GTP]
k
1,k+1·[No]
apply (14, 15).
 |
RESULTS |
Nucleotide Binding Measured by Filtration--
Fig.
1 shows the time progress of nucleotide
binding to isolated UCP. To obtain rates, a low concentration of GTP
(1.2 µM) and 20 mM
Na2SO4, which competes for GTP binding, were
added (10). In all measurements, binding data showed a biphasic fast
and slow increase. The time required for half-maximal binding was ~17
min (Fig. 1A). Similarly, ATP binding was slow (Fig.
1C). However, the nucleoside diphosphates GDP (Fig.
1B) and ADP (Fig. 1D) bound much faster.

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Fig. 1.
Binding of purine nucleotides to isolated
UCP. 2.2 µM UCP was incubated with 1.2 µM 14C-nucleotide (but with 12 µM GTP in the inset in A) in 20 mM Mops, 0.16 mM EDTA, and 20 mM
Na2SO4, pH 6.7, at 4 °C. Binding was
measured with the filter binding method as described under
"Experimental Procedures." The solid lines are
least-square fits according to Equation 8 assuming
Kd = 14 µM.
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|
The measured points (Fig. 1) are best fitted (solid lines)
according to the two-stage binding model. To estimate the dissociation constant (Kd) for the initial loose complex, we made use of the value measured at 0.5 min. For example, the concentration of
bound GTP measured at 0.5 min (the first point) was ~0.11
µM, corresponding to a Kd of 21 µM. The calculated Kd values varied
only between 12 and 28 µM from various measurements. Since these are rough estimates, we fixed Kd at an
average value of 14 µM and performed least-square fitting
for the two parameters Kc
and
k
1 according to Equation 8. Meaningful fitting
can be obtained for the slow binding process measured at 1.2 µM. Thus, for GTP binding (Fig. 1A), the best fit revealed very slow transition rates and a ratio of tight to loose
complex of 30:1 (Table I). GTP binding at
12 µM was much faster (Fig. 1A,
inset) and could be fitted only with uncertainty. Using the
parameters obtained for [GTP] = 1.2 µM (Table I), the theoretical binding calculated for [GTP] = 12 µM fit
the experimental data quite well. Using the same procedure, we
extracted the kinetic parameters for GDP (Fig. 1B) and ATP
(Fig. 1C). Since the transition was faster with ADP (Fig.
1D), only a meaningful distribution constant
(Kc
) could be obtained, and lower limits were estimated for the transition rates. The overall dissociation constants (KD) calculated from the extracted parameters (Table I) are in full agreement with our previous data measured by equilibrium dialysis (8), on anion-exchange method (9), and fluorescence titrations
(10).
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Table I
Kinetic parameters of slow-phase nucleotide binding to isolated UCP
The time course of nucleotide binding was measured by the filter
binding method (Fig. 1) at 4 °C. Fitting for Kc and k 1 was performed according to Equation 8
assuming Kd = 14 µM. The transition
rate constant (k+1) and the overall dissociation
constant (KD) were calculated from
k+1 = k 1 · Kc and KD = Kd/(Kc + 1).
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|
At higher pH (Fig. 2A), GTP
binding appeared to be faster, but weaker with a drastic decrease in
the tight complex population (Table I). We also compared GTP binding
obtained at 4 and 14.5 °C to illustrate the strong temperature
dependence (Fig. 2A). A best fit for the data points at
14.5 °C required Kc
= 25.3 and
k
1 = 1.0 × 10
3
s
1. The data suggest that temperature did not affect the
tight/loose complex distribution (Kc
), but the
transition rates at 14.5 °C were nearly 10-fold faster than at
4 °C.

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Fig. 2.
GTP binding to isolated UCP is dependent on
pH and temperature. 2.2 µM UCP was incubated with
1.2 µM [14C]GTP at pH 6.7, 7.4, and 7.8 and
4 °C (A) and at pH 6.7 and 4 °C and 14.5 °C.
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|
To further evaluate the time-dependent nucleotide binding,
we studied the nucleotide concentration dependence. Fig.
3 summarizes in mass action plots the GTP
binding data measured after incubation for 3, 20, and 180 min. We
calculated the concentrations of bound (UN + U*N) and free GTP
according to Equation 9 using the parameters given in Table I. For pH
6.7 (Fig. 3A), the calculated mass action plots (solid
lines) are straight lines of varying slopes, with KD = 12.9, 3.8, and 1.0 µM for 3, 20, and 180 min, respectively. At pH 7.4 (Fig. 3B), GTP binding
was weaker, with KD values 2-4-fold higher than at
pH 6.7. GTP binding at pH 7.8 was rather weak, with 3-16-fold higher
KD values. In all cases, the theoretical
calculations give good fits to the data points, although the latter
scattered strongly at pH 7.8 (Fig. 3C).

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Fig. 3.
Mass action plots of GTP binding to isolated
UCP. UCP (8-10 µM) was incubated with 0.5-15
µM [14C]GTP at the indicated pH values and
4 °C for 3, 20, and 180 min. The solid lines were
computed according to Equation 9.
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|
To verify the validity of the filtration method, we employed two
approaches to measure the dissociation rate. First, UCP was incubated
(23 °C) with [14C]GTP until equilibrium was reached,
and excess unlabeled GTP was added to displace the prebound
[14C]GTP. As shown in Fig.
4A, the concentration of
prebound [14C]GTP decreased exponentially. Best fits of
the data for pH 6.7 and 7.4 yielded k
1 = 0.44 × 10
3 and 1.5 × 10
3
s
1, respectively. The time course of free
[14C]GTP released into the supernatant followed an
exponential increase with rate constants of 0.98 × 10
3 and 1.9 × 10
3 s
1,
respectively. While the two sets of dissociation rate constants agree,
they are comparable to those extracted directly from the binding data
(Table I) when temperature dependence is taken into account.

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Fig. 4.
Dissociation of [14C]GTP from
UCP. A, displacement experiment. 5.2 µM UCP
was incubated first with 4.7 µM [14C]GTP at
pH 6.7 and 7.4 and 23 °C for 30 min. Then 59 µM
unlabeled GTP was added to displace the prebound
[14C]GTP. B, dilution experiment. 12.8 µM UCP was incubated with 13.1 µM
[14C]GTP at pH 6.7 and 23 °C. The mixture was
concentrated 5.5-fold. After equilibrium (30 min), the mixture was
diluted 21-fold, and nucleotide binding was measured with the filter
binding method. The solid lines are best fits with single
exponential equations.
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|
In the second approach, we measured the equilibrium shift upon
dilution. Before dilution, the bound and free [14C]GTP
concentrations were 42.6 and 5.35 µM, respectively. After a 21-fold dilution, the initial bound and free [14C]GTP
concentrations should be 2.03 and 0.25 µM. The measured values were 1.67 and 0.40 µM, respectively (Fig.
4B). The rapid drop in the complex concentration upon
dilution revealed the presence of a fast dissociating loose complex
(<18%) and a tight complex (>82%), which gave the slow phase of
dissociation with a rate constant of 1.1 × 10
3
s
1, in fair agreement with the rate determined above by
displacement with excess unlabeled GTP.
Anions were known previously to be competitive inhibitors of nucleotide
binding (8, 10). Here we studied the effects of anions on the two
complexes. The influence of anion on nucleotide binding and
dissociation is illustrated with Na2SO4. With
increasing [SO42
] in the incubation
media, the GTP binding curve was shifted progressively downward (Fig.
5A). The anion effect can be
explained in that SO42
can
competitively bind to the nucleotide-binding site and increase the
Kd for the initial loose complex (10). Theoretical fitting using data of Table I gave Kd values of 3.8, 9.4, and 32.7 µM at 2.1, 10, and 48 mM
SO42
, respectively. Attempts to fit
the data with constant Kd but varying
Kc
or k
1 failed,
suggesting that SO42
does not
influence the subsequent conformational change step. Such a finding was
also reported for ATP or ADP binding to myosin (16). A plot of
Kd against [SO42
]
gives a straight line (Fig. 5A, inset), yielding
an intrinsic dissociation constant (Kdo) in
the absence of any anions of 2.5 ± 0.6 µM and an
SO42
inhibition constant
(Ki) of 4.0 mM. The
SO42
inhibition constant measured with
this method is close to the value (1.0 mM) determined
previously by fluorescence titrations (10). Indeed, when
[14C]GTP dissociation induced by excess unlabeled GTP was
followed in the presence of SO42
(Fig.
5B), little difference was observed in the dissociation rate
with 2.1 or 48 mM Na2SO4.

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Fig. 5.
Influence of
SO42 on GTP binding to isolated UCP.
A, 3.6 µM UCP was incubated with 6 µM [14C]GTP in 20 mM Mops and
0.16 mM EDTA containing 2.1-48 mM
Na2SO4, pH 6.7, at 4 °C. The solid
lines are best fits performed according to Equation 8 assuming
Kc = 30.2 and k 1 = 0.1 × 10 3 s 1. Inset, a
plot of apparent Kd versus added
Na2SO4 concentration and fit according to
Kd = Kdo/Ki·[SO42 ] + Kdo, where Kdo
is the intrinsic dissociation constant for the loose complex and
Ki is the inhibition constant of
SO42 . B, the dissociation
of [14C]GTP in the presence of
Na2SO4 was induced by excess unlabeled GTP.
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|
We also measured GTP binding to intact mitochondria from brown adipose
tissue with the filter binding method in parallel with the
centrifugation method (data not shown). The two measurements yielded
the same results. Best fits of the binding data revealed Kc
= 123, suggesting 4-fold higher tight complex
formation with UCP in the mitochondria than in the isolated state.
While at 4 °C the transition rate (k+1) was
comparable to that for the isolated protein, the dissociation rate
(k
1 = 0.033 × 10
3
s
1) was nearly 3-fold slower. The transition rates were
8-fold faster at 23 °C than at 4 °C, similar to the data with the
isolated protein.
Kinetics of Nucleotide Binding Measured by Fluorescence
Methods--
The slow-phase kinetics were also measured by competition
with fluorescent nucleotides as follows. Dansyl-ADP was shown
previously to bind to and dissociate from UCP rapidly (10). The
displacement of prebound dansyl-ADP by nucleotides was used to record
their transition rates (see "Experimental Procedures"). When
experiments were performed with various GTP concentrations (1-8
µM), the fluorescence decrease could be fit with the same
rate constant, suggesting that rate limitation indeed applies. The rate
of fluorescence decrease depends on the added nucleotide (Fig.
6). While GTP and ATP caused a slow
fluorescence increase, GDP and ADP elicited a faster fluorescence
response, with ADP being even faster than GDP. The decreases in
fluorescence can be well fit (solid lines) with a single
exponential equation (Table II).

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Fig. 6.
Slow conformational transition to the tight
complex as measured by competition with dansyl-ADP. A, 0.2 µM UCP was preincubated with 14 µM
dansyl-ADP in 20 mM Mes, pH 6.7, at 14.5 °C. The
fluorescence reached its maximum rapidly and was stable within the
recording time (upper trace). The concentration of the
competing nucleotide (ATP, ADP, GTP, or GDP) added was 4 µM. B, shown are the best fits of the
fluorescence data with a single exponential equation.
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Table II
Rate constants of slow-phase nucleotide binding to isolated UCP at pH
6.7 and 14.5 °C measured by the fluorescence competition method
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To measure the dissociation rate of the tight U*N complex, excess
dansyl-GTP was added to the preformed UCP-nucleotide complex. As shown
in Fig. 7, the rate of fluorescence
increase depends on the competing nucleotide species. It was slow with
GTP and ATP, but faster with GDP and ADP. The rate constants
(k
1) were obtained by fitting and are given in
Table II.

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Fig. 7.
Slow dissociation of nucleotides from UCP as
measured by competition with dansyl-GTP. A, 0.2 µM UCP was first incubated with excess nucleotide (2 µM) for 30 min at pH 6.7 and 14.5 °C. Then a large
excess of dansyl-GTP (20 µM) was added. In control experiments, UCP was first incubated with excess ATP (1.5 mM), and the fluorescence upon addition of dansyl-GTP was
recorded (lower traces). B, shown are the best
fits of the fluorescence data with a single exponential equation.
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|
 |
DISCUSSION |
Nucleotide binding to UCP was found to follow a two-stage process,
with an initial fast but loose binding state and a slow transition into
a tight binding state. In our previous equilibrium experiments (10),
the loose and tight complexes were verified by comparing the binding
data measured by an anion-exchange method and fluorescence titrations.
Most important, the inhibition of H+ transport was shown to
require the tight binding state. Furthermore, the increased resistance
to tryptic digestion of the tight complex witnesses that a major
conformational change is involved (11).
Here we studied the time course of the total binding by a filter
binding method that revealed a clearly biphasic binding process. Although the initial fast phase of binding could not be resolved with
this method, we assume that the rate would be the same
(kon = 3.6 × 105
M
1 s
1) as previously determined
for the dansylated nucleotides by a stopped-flow method (10). The
further slow increase in binding was attributed to the subsequent
transition to the tight complex. The existence of loose and tight
complexes was also demonstrated in dilution experiments (Fig.
4B). The biphasic kinetics can be best described by the
two-stage binding model. Furthermore, the time-dependent
change of the mass action plots could be quantitatively fitted by this
model, thus providing experimental support in two dimensions of time
and GTP concentration.
The two-stage time progress of binding is simulated by theoretical
computations to further illustrate the biphasic binding model. For this
purpose, we fixed the dissociation constant for the initial loose
complex (Kd = 14 µM) and the
tight-to-loose transition rate constant (k
1 = 0.1 × 10
3 s
1) (Table I), but varied
the tight/loose distribution constant (Kc
) (Fig.
8A). The total binding (UN + U*N) is clearly very sensitive to Kc
, as was shown
by the pH dependence (Fig. 2A). Increasing the
Kd at a constant Kc
shifts the
binding curve downward without altering the curvature (Fig.
8B), in line with the effects of anions (Fig. 5). An
increase in the transition rates (k+1 and
k
1) enhances the curvature without affecting the end
point (Fig. 8C), in agreement with the faster binding at
elevated temperatures (Fig. 2B). The sensitivity of these
parameters inherent to the two-stage binding model has allowed an
unambiguous extraction of the kinetic parameters. In Fig.
8D, we calculated the time course of the various UCP-binding species using the parameters given in Table I. The free UCP
concentration (U) decreases exponentially as binding proceeds. The
loose complex (UN) forms instantly (here 0.5 µM at time
0), but its concentration decreases as the conformational change
occurs. The increase in the tight complex (U*N) follows an exponential
progression course from time 0. Since the filter binding method
measures both the loose and tight complexes, the observed binding (UN + U*N) increases exponentially from 0.5 µM, thus showing a
biphasic time course of nucleotide binding.

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Fig. 8.
Theoretical modulations of the two-stage
binding model. Theoretical curves were computed for binding of 1.2 µM GTP to 2.2 µM UCP at pH 6.7 and 4 °C
according to Equation 8 by varying Kc assuming
Kd = 14 µM and
k 1 = 0.10 × 10 3
s 1 (A), by varying Kd
assuming Kc = 30.2 and k 1 = 0.10 × 10 3 s 1 (B), and
by varying k 1 assuming Kd = 14 µM and Kc = 30.2 (C).
Also shown (D) is the time course of concentration variation
for free UCP (U), the loose UCP-nucleotide complex
(UN), and the tight complex (U*N) when 5 µM GTP was added to 2.2 µM UCP at pH 6.7 and 4 °C.
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As shown in Table I, the tight/loose distribution varies moderately
among the four nucleotides so that at equilibrium (pH 6.7 and 4 °C),
the tight complex (U*N) accounts for 71-97% of the total
UCP-nucleotide complex. With increasing pH, the transition rate
constant (k+1) for GTP varied only 2-fold,
whereas the dissociation rate constant (k
1)
increased 5-fold at pH 7.4 and nearly 30-fold at pH 7.8. The
Kc
decreased by ~10- and 43-fold, respectively,
reflecting a strong decrease in the tight complex at equilibrium. The
overall affinity declines accordingly due primarily to a decrease in
Kc
, as shown by 8- and 18-fold increases in
KD, in agreement with the pH dependence reported
earlier (9).
It is interesting that although the initial rapid binding rate
(kon) does not depend on the nucleotide species
(10), the subsequent transition step as measured here is strongly
nucleotide-dependent. With ATP or GTP, the transition is
surprisingly ~45 times slower than with GDP or ADP. This feature is
consistent with the faster inhibition of mitochondrial swelling in the
presence of potassium acetate by the diphosphates (17). Obviously, the
initial fast binding is associated with a less specific protein-ligand
recognition, whereas stronger and thus more specific interactions are
realized by the subsequent slow rearrangement of the binding site. The unusually slow rate is indicative of an intriguingly coordinated rearrangement required to reach the tight binding state and thus ensure
inhibition of H+ transport. The difference in the
dissociation rates (k
1) is even more striking.
ATP dissociates 6-fold faster than GTP, whereas with the diphosphates
GDP and ADP, the rate is 88- and >300-fold faster, respectively. The
even slower transition for nucleoside triphosphates can be interpreted
in terms of a further interaction with the histidine whereby the
binding center has to be deepened (10).
The binding of GTP to brown adipose tissue mitochondria is even slower
than to isolated UCP. This slow binding was associated with a much
weaker initial affinity (Kd
120 µM), presumably due to competition with endogenous ATP
carried over from the cytosol (17). Indeed, when the mitochondria were
washed at higher pH (8.8) prior to the measurements, GTP binding was
found to be faster with a higher initial affinity (data not shown).
A comparison of nucleotides with dansylated nucleotide derivatives
reveals that dansylation accelerates the conformational transition
step. The k+1 increases ~45-, 30-, and 4-fold for GTP, ATP, and ADP, respectively, whereas the increase in
k
1 is more drastic, amounting to ~700-,
240-, and 40-fold, respectively. As a result, a lower tight complex
population was measured for the dansylated nucleotides (11).
Comparison with Other Nucleotide-binding Proteins--
In several
nucleotide-utilizing enzymes, purine nucleotide binding (with
Mg2+) was found to occur in at least two stages, with an
initial loose but rapid association followed by conformational
transitions. The rate of transition in most enzymes was fast,
e.g. 400 s
1 for myosin (16), ~200
s
1 for elongation factor Tu (18), and 4-17
s
1 for p21Ha-ras (19). This differs
largely from the unusually slow transition observed for nucleotide
binding to UCP, presumably due to the fact that in UCP we have the rare
case of free nucleotide (NDP3
and NTP4
)
binding, whereas in the cases mentioned, the magnesium complexes (e.g. (NTP·Mg)2
) interact with the protein.
The interaction of three or four negative charges with the protein can
be expected to be stronger and thus result in larger rearrangements
than in the common case of magnesium-nucleotide complex binding.
The dissociation of prebound nucleotide from these enzymes was reported
to be rather slow. The rate constants for dissociation of ADP and ATP
from G-actin were 3.3 × 10
3 and 5 × 10
4 s
1, respectively (20). ADP dissociation
from the platelet ADP receptor was found to be 2.4 × 10
2 s
1 (21). The dissociation rate in
myosin was very temperature-sensitive (22), increasing from 0.07 to 1.4 s
1 as the temperature increased from 5 to 21 °C. The
G-protein family also had a high affinity for GTP and GDP. The
nucleotides dissociate from the Ras-related nuclear G-protein slowly,
with rate constants of 1.2 × 10
5 and 1.4 × 10
5 s
1, respectively (23, 24). Slow
dissociation (0.85 × 10
3 s
1 for GDP
and 5 × 10
3 s
1 for GTP) was observed
also for elongation factor Tu from Thermus thermophilus
(18). The slow dissociation in these proteins may suggest a common
occluded state of nucleotide in the binding pocket following
conformational rearrangement in the protein.