From the National Laboratory of Biomacromolecules, Institute of
Biophysics, Academia Sinica, Beijing 100101, China
The time course of
8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase
(AK) is a biphasic process. The burst phase ends in the dead-time of
the stopped-flow apparatus (about 15 ms), whereas the slow phase
completes in about 10 min. A Job's plot tests of the binding
stoichiometry demonstrates that there is one ANS binding site on AK,
but only about 70% of the enzyme can rapidly bind with ANS, indicating
that the conformation of native AK molecules is not homogeneous.
Further kinetic analysis shows that the effects of ANS and substrates
concentration on the burst and slow phase fluorescence building agree
well with the multiple native forms mechanism. One form (denoted
N1) binds with ANS, whereas the other (denoted
N2) does not. ANS binding to N1 results in a
burst phase fluorescence increase, followed by the interconversion of
N2 to N1, to give the slow phase ANS binding.
Under urea denaturation conditions, N2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N1 is relatively resistant to denaturation and
unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of
the enzyme catalytic mechanism.
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INTRODUCTION |
It has been accepted that a globular protein in its native state
adopts a single, well defined conformation. However, this concept has
been challenged by several reports that some proteins may exist in more
than one distinct folded form in equilibrium. Evidence for
distinguishing multiple native forms of staphylococcal nuclease has
come from electrophoretic and NMR studies (1-6). For calbindin
D9K, evidence of multiple forms came not only from NMR
studies, but also from x-ray crystal structure (7, 8).
Adenylate kinase (AK1; EC
2.7.4.3) catalyzes the phosphoryl transfer reaction: MgATP + AMP
MgADP + ADP (9, 10). The enzyme contains two distinct nucleotide
binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP
site, which is specific for AMP and uncomplexed ADP. The
substrate-induced conformation changes in AK have been the subject of a
number of investigations (11-22). Based on the comparison of AK
crystal structures representing the enzyme in different ligand
forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef
heart mitochondrial matrix), and enzyme-AP5A complex (from
Escherichia coli), Schulz and co-workers (15, 18) suggested
that AK undergoes large structural changes upon substrates binding.
These conformational changes can be subdivided into two steps; the
first change, corresponding to binding to AMP, only involves the
displacement of the small
-helical domain (residues 30-59 in
E. coli AK), whereas the second change, occurring with
additional binding of substrate ATP, mainly involves the displacement
of the LID domain (residues 122-159 in E. coli AK). However, it is not clear whether the enzyme achieves its catalytic conformation only upon substrate binding or whether the above conformation is an intrinsic property (15). In other words, is there
more than one distinct folded native form of AK in the absence of the
substrate in solution?
Two crystalline forms of adenylate kinase from pig muscle have been
reported, which can be interconverted into each other depending on the
pH of the medium. The conformational change from form A to B is
believed to be an intrinsic property of the enzyme (22, 23). Russell
et al. (14) reported that the higher apparent Mr values of rabbit muscle adenylate kinase
determined from gel filtration data obtained in the presence of DTT,
higher pH, and higher substrate concentration were due to the
conformational changes due to alterations of the intramolecular charge
distribution induced by pH, DTT, and the substrates. Sinev et
al. (21) performed measurements of time-resolved dynamic
radiation-less energy transfer of mutant E. coli AK in which
the solvent-accessible residues valine 169 and alanine 55 were replaced
by tryptophan (the donor of excitation energy), whereas the cysteine
was labeled with either 5- or 4-acetamidosalicylic acid (the acceptor),
respectively. The experimental results confirmed the stepwise manner of
the domain closure of the enzyme upon binding substrates and revealed the presence of multiple conformations of E. coli AK in
solution. However, despite numerous studies using 1H NMR on
AK with different ligands, AMP, ADP, ATP, and AP5A (13, 24-27), the information on the solution structures is limited due to
the relatively large size of the enzyme.
In a previous study from this laboratory (28), it was found that the
time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to AK
was a biphasic process. The burst phase ended in the dead-time of the
stopped-flow apparatus (about 15 ms), whereas the slow phase completed
in about 10 min. The results were interpreted to indicate either
conformational changes during AK binding with ANS or the existence of
multiple forms of the enzyme. In the present study, a kinetic approach
was used to explore the mechanisms of the fluorescence building. The
results clearly reveal the existence of at least two native forms of AK
in equilibrium in solution.
 |
EXPERIMENTAL PROCEDURES |
Reagents
Glucose-6-phosphate dehydrogenase, hexokinase, NADP, ADP, and
ANS were Sigma products; urea was from Nacalal Tesque Inc. (Japan); and
other reagents were local products of analytical grade. Urea solution
was always freshly prepared.
Preparation and Activity Assay of Adenylate Kinase
The enzyme was prepared essentially according to Zhang et
al. (29). The yield was usually about 60 mg of pure enzyme/kg of
rabbit muscle. The final preparation usually had a specific activity
greater than 1600 units/mg and showed only a single peak in SDS
electrophoresis, gel filtration, and reversed-phase FPLC. One unit is
defined as 1 µmol of ATP generated from ADP/min.
The activity assay was made by following the reduction of NADP in a
coupled enzyme solution with hexokinase and glucose-6-phosphate dehydrogenase. The reaction mixture contained 2.5 mM ADP,
2.1 mM Mg acetate, 6.7 mM NADP, 20 units
hexokinase, and 10 units glucose-6-phosphate dehydrogenase in 50 mM Tris-HCl buffer (pH 8.1) The concentration of AK was
determined by the absorption at 280 nm with
A1cm1% = 5.2.
Methods
Jasco-720 and Jasco-500A spectropolarimeters were used for CD
measurements in the far ultraviolet range from 200 to 250 nm. For the
time course of unfolding, the ellipticity was followed at 222 nm for
the changes in the helix content. UV absorbance at 287 nm was measured
with a DU-7500 UV spectrophotometer for steady state and with a Carry
219 spectrophotometer for the time course of unfolding. The data were
obtained after subtracting the base line under the same conditions.
The time course of ANS binding to AK fluorescence was measured using a
SPF-17 stopped-flow system with a syringe ratio of 1:1 (Applied
Photophysics Ltd.). One syringe contains a fixed concentration of ANS
in 50 mM Tris/HCl, 1 mM EDTA at pH 8.1, 25 °C, whereas the other syringe contains the appropriate protein
sample. The concentration of adenylate kinase was 5.0 µM
for all experiments unless otherwise specified. The concentration of
ANS was determined using the extinction coefficient
360 nm = 4.95 × 103
M
1 cm
1 in aqueous solution. The
samples were excited at 378 nm, and the emission above 410 nm was
detected using a wavelength cut-off filter. The dead-time in these
experiments was about 15 ms. In all measurements, the base line was
calibrated with ANS solution.
Digitized kinetic traces were analyzed using the software provided with
the Applied Photophysics Kinetic Spectrometer Workstation (Archimedes).
Apparent observed first-order rate constants
(kobs) were obtained by nonlinear least-squares
fits of the data to Equation 1.
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(Eq. 1)
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At is the total fluorescence intensity
measured at time t, Af is the amplitude of the burst
phase reaction, As is the amplitude of the slow
phase reaction, and kobs is the observed rate
constant of the slow phase reaction.
Kinetics
Two possible mechanisms can be proposed to interpret the
biphasic fluorescence increase of ANS binding to AK. 1) The rapid formation of AK·ANS complex is followed by a slow conformational transformation to result in the slow phase reaction (this is designated as a conformational transformation mechanism). 2) At least two native
forms of AK exist in solution, of which one binds rapidly with ANS and
the other does not (designated as a multiple forms mechanism). These
two mechanisms can be distinguished by kinetic analysis.
Conformational Transformation Mechanism--
The conformational
transformation mechanism can be expressed by Reaction 1.
AK and ANS first rapidly complex to form an intermediate
([AK·ANS]), resulting in the burst phase fluorescence. This
intermediate then undergoes a conformational adjustment to give
the slow phase fluorescence increase. If this mechanism is correct, the
fraction of burst and slow phases should remain constant with
varying ANS concentration.
Multiple Forms Mechanism--
Consider that there are two native
forms of AK in equilibrium in solution, as shown in Reaction 2. One
form (denoted N1) binds with ANS, whereas the other
(denoted N2) does not. ANS binding to N1 makes
the burst phase fluorescence, as shown in Reaction 3. N2 is
then converted to N1 and binds with ANS to give the slow phase fluorescence increase, as shown in Reaction 4.
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(Eq. 2)
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{smtext}and
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(Eq. 3)
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Here, [N1]0,
[N2]0, and [N]0 are the
equilibrated concentration of forms N1, N2, and
total enzyme, respectively. The kinetics of ANS binding to AK are based
on the following assumptions. 1) Steady-state conditions are
instantaneously reached between N1, ANS, and
N1·ANS and also between N1, ATP, and
N1·ATP, and the interconversion of N2 to
N1 is the rate-limiting step. 2) Only N1 binds
with ANS and ATP. The first assumption is justified because the enzyme
binding with substrates or substrate analogs is usually a very fast
reaction. The second assumption seems also to be justified because
experiments of soaking of crystals with ANS or ATP revealed that
crystalline form B of AK binds with ANS or ATP (12). Under the above
conditions, we obtain the following equations.
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(Eq. 4)
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(Eq. 5)
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(Eq. 6)
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(Eq. 7)
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Substituting Equations 4-6 into Equation 7 gives Equation 8.
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(Eq. 8)
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(Eq. 9)
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where
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(Eq. 10)
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and
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(Eq. 11)
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Integrating Equation 9, under the boundary conditions that at
t = 0, [N2] = [N2]0, and that at t = t, [N2] = [N2], gives Equations
12 and 13.
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(Eq. 12)
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(Eq. 13)
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The observed slow phase fluorescence building rate constant
(kobs) is shown below.
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(Eq. 14)
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(Eq. 15)
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Substitute A and B into Equation 15 to
give Equation 16.
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(Eq. 16)
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Here K1 =
k
1/k1. The amplitude of the
total fluorescence (Ft) is shown in Equation 17.
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(Eq. 17)
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At t = 0,
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(Eq. 18)
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The amplitude of the burst phase fluorescence
(Ff) is given by Equation 19.
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(Eq. 19)
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In the absence of substrates, Equations 14, 15, 17, 20, and 21
can be written as shown below.
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(Eq. 20)
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(Eq. 21)
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(Eq. 22)
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(Eq. 23)
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(Eq. 24)
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(Eq. 25)
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The fractions of the burst
(Ff/Ft) and slow phase
(Fs/Ft) are shown in Equations 26
and 27.
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(Eq. 26)
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(Eq. 27)
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 |
RESULTS |
Time Course of ANS Binding to AK--
It has been reported that
there is one ANS binding site on native AK close to the site of ATP
binding (30) and the binding of AK with ANS can raise the quantum yield
of ANS fluorescence as well as shift the emission peak to lower
wavelengths. Fig. 1 shows the time course
of fluorescence increase by ANS binding to AK at pH 8.1 in the presence
of DTT. The fluorescence increase is obviously a biphasic process. The
burst phase completes within the dead-time of mixing in the
stopped-flow apparatus, and the slow phase ends in about 10 min.

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Fig. 1.
Time course of ANS binding to AK. The
final enzyme concentration was 5 µM, and the ANS
concentration was 0.2 mM, with 50 mM Tris-HCl,
1 mM EDTA, and 1 mM -mercaptoethanol at
25 °C, pH 8.1.
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Stoichiometry of ANS Binding to AK--
To check the stoichiometry
of ANS binding, the fluorescence increase for both the burst and the
total increase were treated by the method of Job (31). The results are
shown in Fig. 2. The maximum intensity of
the total fluorescence increase is at a 1:1 molar ratio of ANS to AK,
indicating one ANS binding site on AK. However the burst fluorescence
intensity reaches a maximum at the molar ratio 0.4:0.6 of ANS to AK,
indicating only about 70% of the enzyme can rapidly bind with ANS. The
above results suggest that the conformation of AK molecules in solution
is not homogeneous.

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Fig. 2.
Job's plot for ANS binding to AK. The
fluorescence was measured at 378 nm excitation, 478 nm emission, The
total concentration of enzyme and ANS was 25 µM in 50 mM Tris-HCl buffer, pH 8.1, at 25 °C containing 1 mM EDTA. Open circles, total fluorescence; filled circles, burst phase fluorescence.
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Effects of ANS Concentration on ANS Binding to AK--
The effect
of ANS concentration on the kinetics of ANS binding to AK was explored
by measuring the burst and total fluorescence increase of a series of
solutions containing increasing concentrations of ANS. Fig.
3A shows the amplitudes of the
burst phase (Ff) and total (Ft)
fluorescence increase as functions of the ANS concentration at pH 8.1. The inset in Fig. 3A shows the fractions of the
burst (Ff/Ft) and slow phases (Fs/Ft) as functions of ANS
concentration, showing that Ff/Ft
decreases and Fs/Ft increases
with increasing ANS concentration and that both gradually approach a
constant value at higher concentrations of ANS. Similar results were
obtained at pH 6.0 and pH 9.0 (data not shown).

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Fig. 3.
Effect of ANS concentration on ANS binding to
AK. A, changes of ANS binding fluorescence versus
ANS concentration (open circle, total fluorescence;
filled circle, burst phase). Inset plot shows the
fraction of burst (Ff/Ft,
triangle) and slow phase
(Fs/Ft, circle)
versus the concentration of ANS, and the solid
lines are calculated using Equations 26 and 27 with
K1 = 2.1 and K2 = 37 mM. B, double-reciprocal plot of the ANS
concentration versus fluorescence intensity (open
circles, total fluorescence; filled circles, burst
phase). AK concentration was 5 µM; buffer solution is 50 mM Tris-HCl, containing 1 mM EDTA and 1 mM -mercaptoethanol at pH 8.1 and 25 °C.
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According to Equations 26 and 27, when [ANS]
0, then
Ff/Ft
1 and
Fs/Ft
0; when [ANS]
, then Ff/Ft
K1/(1 + K1),
Fs/Ft
1/(1 + K1), and
Fs/Ff = 1/K1. The equilibrium values ratio of
Fs/Ff is 0.47, equivalent to a
K1 value of 2.1. The solid lines in
the inset plot of Fig. 3A are from calculated
values using Equations 26 and 27 with K1 = 2.1 and K2 = 37 mM; these values agree
well with the experimental results.
Fig. 3B shows the double-reciprocal plot of
1/Ff and 1/Ft versus
1/[ANS]; both give straight lines with the same slope. The kinetic
parameters obtained by fitting the data with Equations 24 and 25 are
listed in Table I.
Fig. 4 shows that the observed
first-order rate constant (kobs) of the slow
phase fluorescence change decreases with increasing ANS concentration.
The interconversion rate constants k1 and
k
1 can be obtained by fitting the date with
Equation 20. According to Equation 20, when [ANS]
0, kobs
k
1 + k1, and when [ANS]
,
kobs
k
1. From the
data in Fig. 4, k
1 can be estimated to a
little less than 0.006. Equation 20 has three unknowns:
k1, k
1, and
K2. A nonlinear fit can be avoided by rewriting
Equation 20 as shown in Equation 28.
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(Eq. 28)
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When k
1 is chosen properly, a plot of
1/(kobs
k
1)
versus [ANS] yields a straight line. When
k
1 is chosen too high, the curve bends upward,
and, when chosen too low, downward. As can be seen from the
inset in Fig. 4, a good fit is obtained using
k
1 = 0.0055 s
1, which yields
k1 = 0.013 s
1 and
K2 = 55 µM.

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Fig. 4.
Observed slow phase ANS fluorescence building
rate constant versus ANS concentration. Inset,
same data plotted as 1/(kobs k 1) (see text) versus the ANS
concentration. The experimental conditions were the same as in Fig.
3.
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Effect of AK Concentration on ANS Binding to AK--
Fig.
5 shows the effect of AK concentration on
the amplitudes of the burst and slow phase fluorescence. Both increase
linearly with increasing concentration of AK. This result indicates
that there is no dimer or aggregation formation in this enzyme
concentration range.

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Fig. 5.
Effect of AK concentration on ANS binding to
AK. Triangles, burst phase; circles, total
change. The concentration of ANS was 0.2 mM containing 50 mM Tris-HCl, 1 mM EDTA, and 1 mM -mercaptoethanol at 25 °C.
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Substrate Effects on the Kinetics of ANS Binding to AK--
The
overall effects of substrates on the kinetics of ANS binding to AK were
also investigated. Specified concentrations of substrates were first
added to ANS solution, then mixed 1:1 with AK solution by stopped-flow.
Fig. 6 shows that the substrates and
their analogue, AMP, ADP, ATP, and AP5A, inhibit both the burst (Fig. 6A) and slow phase (Fig. 6B)
fluorescence building. The inhibition efficiency is in the order:
AMP < ADP < ATP < AP5A. Further
experiments were carried out to study the effects of ATP and MgATP
concentration on the kinetics of ANS binding to AK. Fig.
7 shows 1/Ff
(A) and 1/Ft (B)
versus 1/[ANS] for various ATP concentrations. Fig.
8 shows 1/Ff
(A) and 1/Ft (B)
versus 1/[ANS] for various MgATP concentrations. The
results clearly show that ATP and MgATP act as competitive inhibitors
for ANS binding to AK.

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Fig. 6.
Effect of nucleotide substrates on the
binding of ANS to AK. A, burst phase; B, slow
phase. The enzyme (10 µM) was mixed with various
concentrations of substrates containing 200 µM ANS in 50 mM Tris-HCl buffer containing 1 mM EDTA at pH
8.1. The fluorescence was measured at 25 °C with 378 nm excitation and 478 nm emission.
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Fig. 7.
Effect of ATP concentration on the kinetics
of ANS binding to AK. A, 1/Ff
versus 1/[ANS] with varying ATP. B,
1/Ft versus 1/[ANS] with varying ATP.
The experimental conditions were the same as in Fig. 6. The
concentrations of ATP are: 1, 0.0 mM;
2, 4 mM; 3, 8 mM;
4, 12 mM; 5, 16 mM;
6, 20 mM.
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Fig. 8.
Effect of MgATP concentration on the kinetics
of ANS binding to AK. A, 1/Ff
versus 1/[ANS] with varying MgATP. B,
1/Ft versus 1/[ANS] with varying MgATP.
The experimental conditions were the same as in Fig. 6. The
concentrations of MgATP are: 1, 0.0 mM;
2, 4 mM; 3, 8 mM;
4, 12 mM; 5, 16 mM;
6, 20 mM.
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Effect of pH on ANS Binding to AK--
The dependence of the burst
and slow phase fluorescence building on pH (Fig.
9) illustrates that the fraction of the
slow phase increases but that of the burst phase decreases with
increasing pH in the pH range from 6 to 9 in the presence of DTT or
-mercaptoethanol. At pH 8.1 in the absence of either DTT or
-mercaptoethanol, the AK binding ANS fluorescence shows the same
kinetics behavior as in the presence of DTT or
-mercaptoethanol
except that the amplitude of the slow phase reaction increases to 0.43. Similar results were also obtained at other pH values (data not
shown).

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Fig. 9.
Effect of pH on the ANS binding
fluorescence. The enzyme concentration was 5 µM
containing 1 mM EDTA and 1 mM
-mercaptoethanol (circle) or 1 mM DTT
(square), or in the absence of -mercaptoethanol/DTT (triangle). Open symbols, fraction of slow phase;
filled symbols, fraction of fast phase. For pH higher than
7, the buffer solution contained 50 mM Tris-HCl. For pH
lower than 7, the buffer solution contained 100 mM sodium
citrate solution.
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Denaturation of Adenylate Kinase in Urea--
Urea unfolding of AK
was studied by measuring the changes of CD at
222 nm
and of the UV absorbance at 287 nm (Fig. 10). Both measurements changed little
up to 1.8 M urea and changed sharply between 1.8 and 2.5 M urea. The concentration of urea required to produce
changes of 50% in either method was about 2.0 M.

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Fig. 10.
Urea denaturation of AK. Filled
circles are the relative change monitored by CD at 222 nm;
open circles are the relative change monitored by UV at 287 nm. The enzyme concentration was 19.5 µM for UV
measurement and 10 µM for CD measurement. The solution contained 50 mM Tris-HCl, 1 mM DTT, and 1 mM EDTA at pH 8.1. All measurements were carried out at
25 °C.
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The unfolding kinetics were followed using the time courses of
ellipticity at 222 nm and absorbance at 287 nm. Fig.
11 shows the kinetics of unfolding,
with the early time history recorded in the inset plot. The
unfolding of the tertiary and secondary structures of the enzyme are
both biphasic processes. The amplitude of slow phase increased with
increasing urea concentration, whereas that of the burst phase was
nearly constant in the range of 2.3-3.4 M urea (Fig.
12). The unfolding rate constant of the
slow phase increased from 0.005 s
1 in 2.3 M
urea to 0.02 s
1 in 3.4 M urea, whereas the
change of the burst phase rate constant was too fast to determine (data
not shown).

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Fig. 11.
Urea denaturation time course of AK.
Denaturation was monitored by UV 287 nm with the final urea
concentration 3 M at 25 °C. The enzyme concentration was
19.5 µM. The inset plot shows the early
behavior. Other conditions were the same as in Fig. 10.
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Fig. 12.
AK unfolding amplitude fraction
versus urea concentration. Open circles are for
burst phase, triangles are for slow phase, and
squares are for total phases monitored by UV at 287 nm. The
corresponding filled symbols represent the values monitored by CD at 222 nm.
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Unfolding Kinetics of the AK·ANS Complex--
The unfolding of
the AK·ANS complex was studied by first incubating 0.2 mg/ml AK with
200 mM ANS for 1 h at 25 °C. This equilibrated solution was then mixed 1:1 with a 5.6 M urea solution
containing 200 µM ANS. The time course of unfolding,
monitored by ellipticity at 222 nm (Fig.
13, line a), shows a
monophasic process with a rate constant of 0.016 s
1. A
control experiment showed that there was no influence of ANS on the CD
spectra of AK (data not shown). For comparison, the unfolding of AK in
the absence of ANS, monitored by ellipticity at 222 nm (Fig. 13,
line b) is clearly a biphasic process.

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Fig. 13.
Urea unfolding kinetics of AK·ANS complex.
Open circles (line b) are for unfolding of AK in
the absence of ANS, filled circles (line a) are
for the unfolding of AK·ANS complex monitored by CD at 222 nm. The
enzyme was 25 µM equilibrated with 0.2 mM ANS. The urea concentration was 2.8 M with the same ANS
concentration.
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 |
DISCUSSION |
The above studies provide evidence to suggest rabbit muscle
adenylate kinase exists in solution in at least two folded forms in
equilibrium. One form (denoted N1) rapidly binds with ANS, whereas the other (denoted N2) does not. N1
binding with ANS causes the burst phase fluorescence increase.
N2 is slowly converted to N1 and then binds
with ANS, resulting in the slow phase of fluorescence increase.
ANS is widely used as a probe of hydrophobic region in protein
structures. The fluorescence intensity of ANS bound to a protein is
dependent on the microenvironment of the binding site (32). Experiments
wherein AK crystals were soaked with ANS or ATP revealed that ANS or
ATP binding occurred only with AK of crystalline form B. ANS occupies
the pocket formed between the
-sheet, loop 16-22, helix 23-30, and
the C-terminal helix. This pocket was originally assigned to the
adenosine moiety of AMP site (12), but has recently been recognized as
the ATP site (33, 34). Compared with the results obtained in the
present study, it appears highly suggestive that N1 relates
to the crystalline form B, and N2 to crystalline form
A.
AK undergoes a domain movement when it binds with substrates or their
analogues. ANS binding to AK should also induce a conformational change
of the enzyme. If the slow phase fluorescence comes from this domain
movement, the fraction of the burst and slow phases should keep
constant with varying ANS concentration (see "Experimental Procedures"). Our observation is that the fraction of the burst phase
decreases and that of the slow phase increases with increasing ANS
concentration, and this is incompatible with the conformational transformation mechanism.
The present experimental results demonstrate that the fraction of
N1 decreases and that of N2 increases at higher
pH, consistent with the study of crystal structures. N1 and
N2 equilibrate with each other in solution. The fractions
of N1 and N2 depend on the solution pH. High
and low pH values favor N2 and N1 respectively consistent with the observation of the prevalence of crystalline forms
A and B at different pH values. The report by Russell et al.
(14) that AK adopts a more open conformation at higher pH in the
presence of 1 mM DTT seems to contradict our observations and crystal studies. This contradiction may arise from the different definitions of the "open" and "closed" forms. The open and
closed forms determined by apparent Mr refer to
the whole size of the enzyme, whereas the open and closed forms
discussed here and in the crystal structure studies refer to the
hydrophobic pocket of the enzyme. According to the data provided by
Sachsenheimer and Schulz (11), the crystal size of form A, which has a
closed hydrophobic pocket, is slightly bigger than that of the form B, which has an open hydrophobic pocket.
Under urea denaturation, the unfolding amplitude of the burst phase is
independent of the urea concentration in the range of 2.3-3.5
M, whereas the amplitude of the slow phase increases at
higher urea concentrations, suggesting the existence of at least two
native forms of AK in solution. The biphasic process of unfolding
cannot be interpreted as the formation of an unfolding intermediate,
because the unfolding amplitude of the burst would not then remain
constant. Perhaps one form of AK is easily perturbed by urea and
unfolds completely at lower concentrations of urea, so that the
unfolding amplitude of the burst phase is independent of the denaturant
concentration. The other form is then relatively resistant to urea
denaturation and its unfolding fraction increases at higher denaturant
concentrations. The fraction of the burst phase of ANS binding
fluorescence is very close to that of the slow phase of the urea
denaturation, and the fraction of the slow phase of ANS binding
fluorescence is very close to that of the burst phase of urea
denaturation. This suggests that the form which unfolds slowly can
rapidly bind with ANS, whereas the other form which unfolds fast can
not. The equilibrium constant K'1 (equilibrium
concentration ratio of N2 to N1, equivalent to
1/K1) can be calculated from either the
unfolding or the ANS binding experiment.
From unfolding amplitudes, we obtain Equation 29.
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(Eq. 29)
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From ANS binding amplitude, we obtain Equation 30.
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(Eq. 30)
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Here N1 denotes the form which unfolds slowly and can
bind with the ANS burst, and N2 denotes the form which
unfolds burst and cannot bind with ANS. The equilibrium constants
calculated from both experiments agree very well and the
interconversion free energy
G0 from
N1 to N2 is calculated according to Equation 31.
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(Eq. 31)
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A previous study reported that the slow phase fluorescence
building can be catalyzed by peptidyl prolyl cis/trans
isomerases (28). The calculated free energy
G0 from N1 to N2 is
close to that of proline isomerization, giving further evidence that
the interconversion of the two forms involves the cis/trans
peptidyl prolyl isomerization of proline residue.
The urea denaturation kinetics of the AK·ANS complex also confirms
the suggestion of at least two native forms of AK in equilibrium in
solution. At equilibrium with 0.2 mM ANS, about 65% of the AK molecules are in the form of N1·ANS complex, 25% in
the form of N1, and 10% are in form of N2.
N1·ANS and N1 unfold fast, whereas the
unfolding of N2 is relatively too small to observe, so the observed unfolding curve is single phase.
From the above discussion, the mechanisms of ANS binding and urea
denaturation of AK can be written as shown below, where U represents
the denatured state of AK.
The multiple forms of AK co-existent in solution may shed some
light on the interpretation of the catalytic mechanism of the enzyme.
One form of AK may fit the substrates MgATP and AMP as well as fit the
inhibitor AP5A, so that AP5A acts as a
competitive inhibitor for the forward reaction, whereas the other form
can fit the substrates MgADP and ADP, but not fit the inhibitor
AP5A, so that AP5A acts as a noncompetitive
inhibitor for the reverse reaction.
In summary, the evidence presented here suggests that at least two
native forms of AK co-existent in solution. One form unfolds slowly and
binds rapidly with ANS, whereas the other form unfolds quickly and does
not bind with ANS. In the absence of substrates, these two folded forms
equilibrate with each other in solution. Changes of the solution pH or
the presence of substrates shifts the equilibrium to fit the new
conditions. Furthermore, interconversion of these two forms may involve
the cis/trans peptidyl prolyl isomerization.
We express our sincere thanks to Professor
C. L. Tsou of the Institute of Biophysics, Academia Sinica, for
many helpful suggestions.