Starting from the E1 state, with high binding affinity for the substrates ions and nucleotides on one side of the membrane, the terminal c-phosphate group of ATP is transiently transferre
Trang 1CopB-B, the catalytic fragment from an archaeal
thermophilic ATP-driven heavy metal transporter
Christian Vo¨llmecke, Carsten Ko¨tting, Klaus Gerwert and Mathias Lu¨bben
Lehrstuhl fu¨r Biophysik, Ruhr-Universita¨t Bochum, Germany
Introduction
The biological role of P-type ATPases is ATP-driven
transport of ions against their concentration gradients
along membranes They form a heterogeneous
super-family, which has been divided into several categories
according to sequence similarity and substrate
specific-ity [1] Among these, the Ca- and Na⁄ K-ATPases
belong to the well-studied class II enzymes Another
large group (class Ib) comprises the so-called
CPX-ATPases, which are responsible for the import or
export of soft metals, such as copper, zinc, silver, lead,
cobalt or cadmium
CPX-ATPases are evolutionarily related and have a
common architecture, consisting of a hydrophobic part
with a predicted eight transmembrane helices, in which
the central ion binding site resides Their peripheral part is extensively hydrophilic and contains several structural and functional modules, such as nucleotide binding (N), phosphorylation (P), actuator (A) and heavy metal binding (HMA) domains
During the catalytic cycle, P-type ATPases, also called E1E2-ATPases, undergo ordered large-scale domain movements, in which ion translocation is coupled to the energy released from ATP hydrolysis Starting from the E1 state, with high binding affinity for the substrates (ions and nucleotides) on one side
of the membrane, the terminal c-phosphate group of ATP is transiently transferred to a conserved aspartic acid, forming a covalently bound aspartyl-phosphate
Keywords
fluorescence spectroscopy;
Fourier-transform infrared spectroscopy;
heavy metal translocation; P-type ATPase;
reaction mechanism
Correspondence
M Lu¨bben, Lehrstuhl fu¨r Biophysik,
Ruhr-Universita¨t Bochum, Universita¨tsstr.
150, D-44780 Bochum, Germany
Fax: +49 234 32 14626
Tel: +49 234 32 24465
E-mail: luebben@bph.rub.de
(Received 14 May 2009, revised 24 July
2009, accepted 21 August 2009)
doi:10.1111/j.1742-4658.2009.07320.x
The mechanism of ATP hydrolysis of a shortened variant of the heavy metal-translocating P-type ATPase CopB of Sulfolobus solfataricus was studied The catalytic fragment, named CopB-B, comprises the nucleotide binding and phosphorylation domains We demonstrated stoichiometric high-affinity binding of one nucleotide to the protein (Kdiss 1–20 lm) Mg
is not necessary for nucleotide association but is essential for the phospha-tase activity Binding and hydrolysis of ATP released photolytically from the caged precursor nitrophenylethyl-ATP was measured at 30C by infra-red spectroscopy, demonstrating that phosphate groups are not involved in nucleotide binding The hydrolytic kinetics was biphasic, and provides evidence for at least one reaction intermediate Modelling of the forward reaction gave rise to three kinetic states connected by two intrinsic rate constants The lower kinetic constant (k1= 4.7· 10)3s)1at 30C) repre-sents the first and rate-limiting reaction, probably reflecting the transition between the open and closed conformations of the domain pair The subse-quent step has a faster rate (k2= 17· 10)3s)1 at 30C), leading to prod-uct formation Although the latter appears to be a single step, it probably comprises several reactions with presently unresolved intermediates Based
on these data, we suggest a model of the hydrolytic mechanism
Abbreviations
cgATP, caged ATP; mant-ATP, 3¢-N-methylanthraniloyl-ATP; AMPPNP, adenosine 5’(b,c-imido)triphosphate.
Trang 2intermediate The phosphorylated E1 state switches
to the phosphorylated E2 state with low affinity for
the substrate ion, which is released to the other side
of the membrane after hydrolysis of the phosphoryl
bond Extensive information about the catalytic
mechanism has been obtained from investigations of
various P-type ATPases [2–5] Many details on the
molecular function and structural models of ground
state and various intermediate states have been
obtained for Ca-ATPase [6], which is regarded as
virtually paradigmatic for the P-type ATPases
Ca-ATPase and Na⁄ K-ATPase have been extensively
investigated by time-resolved FTIR absorbance
differ-ence spectroscopy using various nucleotides and
nucleo-tide analogues [7–13] These studies have suffered from
the fact that the described mammalian proteins could
only be purified from native tissue material The
holo-proteins were difficult to express in Escherichia coli,
which precluded the use of site-directed mutant proteins
or group-specific isotopically labelled proteins for
spec-tral comparisons, which are crucial for assignment of
protein-associated absorbance difference bands
Bacterial CPX-ATPases consist of a single subunit
and can be readily expressed in the heterologous host
Escherichia coli Proteins of this subclass are therefore
suited for site-directed mutagenesis, and would be ideal
candidates for the study of molecular reaction
mecha-nisms However, the 3D structure, which would be
enormously helpful in understanding the molecular
mechanism of CPX-ATPase, is unknown Previously,
various attempts at comparative modelling have created
a structural model of the holoenzyme [14–16] Using
‘divide and conquer’ strategies, the partial 3D structures
of various modules have been determined, such as the
HMA domain of the CPX-ATPases of Listeria
mono-cytogenesand Bacillus subtilis, the N⁄ P and A domains
of Archaeoglobus fulgidus CopA and the N⁄ P domains
of Sulfolobus solfataricus CopB [17–21] In order to
study the reaction mechanism of the ATPase, we
explored here whether a truncated variant of CopB could
act as model for the holoenzyme Therefore, the soluble
catalytic fragment CopB-B, comprising the hydrophilic
N⁄ P domains of CopB from Sulfolobus solfataricus
(Fig 1) was probed The activities of the catalytic
fragment were investigated using enzymological,
fluores-cence [22] and infrared spectroscopy [23] methods
Results
Nucleotide binding to CopB-B
The catalytic fragment N⁄ P, also called CopB-B,
con-sists of the nucleotide binding and phosphorylation
domains of the thermophilic CPX-ATPase CopB from
S solfataricus It was expressed in E coli, crystallized
in a nucleotide-free state, and its structure was deter-mined [21] (see Fig 1) The domains are connected by hinge peptides, which allow substantial flexibility of both domains relative to each other The domains appear to be in a so-called closed orientation, into which the substrate nucleotide, ATP, has been modelled by superposition on the nucleotide-bound structure of Ca-ATPase (Fig 1) The purine moiety fits into a cleft of the nucleotide-binding domain, whereas
Fig 1 3D structural model of the catalytic fragment CopB-B of the heavy metal-translocating CPX-ATPase CopB from Sulfolobus solfa-taricus (PDB code 2IYE) The protein is displayed in half-transparent molecular surface representation, and the conserved phosphoryl-atable Asp416 is shown The adenine nucleotide shown was modelled after structural superposition with the ADP ⁄ AlF 3 -bound structure of Ca-ATPase (PDB code 1WPE).
Trang 3the phosphate groups are located in the vicinity of the
phosphorylation domain It should be taken into
account that our model of the nucleotide-bound state
of CopB-B is relatively crude with respect to the
phos-phate region, and should not be interpreted as
assign-ing possible protein interaction sites to functional
groups of the substrate [21]
The binding interaction of CopB-B with various
adenine nucleotides under stoichiometric conditions
was qualitatively verified by gel filtration of the
nucleo-tide⁄ protein complex and subsequent analysis of the
nucleotides of the collected fractions using
high-perfor-mance liquid chromatography on a reverse-phase
column (see Appendix S1) Equilibrium binding of
nucleotides was quantitatively investigated using the
fluorescent analogue 3¢-N-methylanthraniloyl-ATP
(mant-ATP) (Fig 2) Binding to the protein at
saturat-ing nucleotide concentrations resulted in a 4.5-fold
increase of emission intensity, demonstrating that the
fluorophore becomes positioned in a location that is
less exposed to quenching molecules In addition, the
emission peak shifts from 444 to 434 nm, indicating
that, upon binding, the fluorescent substituent moves
from the hydrophilic solvent into the more
hydropho-bic protein environment (Fig 2A) To assess the
speci-ficity of binding, we displaced the bound mant-ATP
by addition of excess ATP The kinetic dissociation of
the mant-ATP⁄ protein complex appears to be
rela-tively rapid, as the process could not be resolved
within the manual mixing time This reversible ligand
competition shows that the nucleotide portion of the
analogue is responsible for the specific interaction with
the protein
A titration of the nucleotide binding site under
stoi-chiometric conditions (i.e when the molar
concentra-tions of mant-ATP and protein have values much
greater than Kdiss) resulted in a linear increase of
fluo-rescence with ligand addition up to the saturation
point, and above it in constant fluorescence (data not
shown) Extrapolating the lines to their intercept gave a
binding stoichiometry of one nucleotide per CopB-B fragment
For determination of the binding constant Kdiss, the conditions were adjusted such that the concentrations
of mant-ATP and protein were of the same order as the expected Kdiss The hyperbolically shaped titration
A
B
C
Fig 2 Equilibrium binding of CopB-B with nucleotides (A)
Fluores-cence spectra of 0.5 l M mant-ATP in 5 m M Na ⁄ Mes buffer, pH
6.2, at room temperature in the absence (dashed lines) or presence
(continuous lines) of CopB-B in large stoichiometric excess (15 l M ).
(B) Fluorescence titration of 0.5 l M mant-ATP with CopB-B The
fluorescence at emission wavelength 434 nm is given in arbitrary
units; [Et] = total concentration of CopB-B (C) Determination of
ligand dissociation constants from competitive titrations of 0.5 l M
CopB-B with mant-ATP in the presence of the indicated total
con-centrations ([L0]) of ATP (squares), ADP (circles) and AMP
(trian-gles) for determination of the apparent Kdissapp Data were analyzed
according to Eqn (4) The bars indicate K app
diss errors from individual fits of titration curves obtained at fixed competitor concentrations.
Trang 4curve under experimental condition 1 described in the
Experimental procedures (mant-ATP held constant) is
shown in Fig 2B A non-linear regression fit of the
measured data results in a binding constant of 1 lm
according to Eqn (1) The same results were obtained
when titrations were performed under experimental
condition 2 (protein held constant) Nucleotide binding
was highly sensitive to the salt concentration, with the
Kdiss increasing to 40 lm at 100 mm NaCl or
(NH4)2SO4 Notably, binding does not require Mg2+;
the affinity is reduced by a factor of 10 in the presence
of 1 mm MgCl2(Table 1)
The binding specificity of the protein to mant-ATP
can be demonstrated by its displacement by other
nucleotides that are added in slight excess to the
complex It is clear from the displacement of bound
mant-ATP by ATP and related compounds that these
nucleotides interact with the same protein binding
site Ligand competition could thus be exploited for
determination of binding constants of non-fluorescent
nucleotides According to Eqn (4), the apparent
affin-ity Kdissapp of CopB-B for mant-ATP is significantly
increased with higher concentrations of competitor
nucleotide Based on a series of fluorescence titrations
of mant-ATP to CopB-B in the presence of various
competitor concentrations [L0], the binding constant of
the nucleotide can be determined from the slope of
the linear plot of the apparent binding constants Kdissapp
and [L0] With the ligand ATP, a binding constant
Kdisslig of 10 lm was obtained (Fig 2C) The
non-hydro-lysable analogue adenosine 5¢(b,c-imido)triphosphate
(AMPPNP) had binding properties comparable to
those of ATP (Table 1) Structural modification of the
purine moiety had no significant effect, as ATP and
GTP showed affinities in the same order of magnitude
On the other hand, ADP, the product of the ATPase reaction, bound to CopB-B with approximately half of the affinity of ATP AMP had a comparable Kdisslig of approximately 30 lm (Table 1), which indicates that the b- and c-phosphate groups are less important for the binding process than the base⁄ sugar part A remarkable observation is the binding of caged ATP (cgATP) with an affinity similar to that of ATP (Table 1), which was verified independently by HPLC (see Fig S1)
Catalytic activity During catalytic activity, the c-phosphate of ATP is transiently transferred onto the strictly conserved aspartic acid located in the phosphorylation domain, which is Asp416 in CopB-B [21] In the P-type ATPase holoprotein, the A domain comes into contact with the
N⁄ P domain pair, promoting the hydrolysis reaction
by release of inorganic phosphate from the phosphory-lated intermediate state [5] Formation of the phos-phorylated intermediate of CopB-B with the substrate ATP has been shown previously [24], as well as its hydrolytic activity with the artificial substrate p-nitro-phenyl phosphate, even though the A domain is absent
in this construct This is probably due to thermal activation of the phosphatase reaction The catalytic activity using the native substrate Mg-ATP gave approximately five times higher rates, amounting
to 50–70 nmol (mgÆmin))1 Variation of substrate concentration revealed a simple hyperbolic Michaelis– Menten-type dependence and a KM of 1 mm, which reflects relatively poor kinetic substrate affinity compared with the thermodynamic ligand association constant Kdisslig of ATP (Fig 3A) Nevertheless, these relationships are consistent because high substrate con-centrations are needed to overcome the high-affinity binding of the product ADP (Table 1) under kinetic steady-state conditions No production of inorganic phosphate was observed in the absence of Mg2+, which indicates that Mg-ATP is the substrate of CopB-B Furthermore, the ATPase activity increased in the temperature interval between 20–70C (Fig 3B) At higher incubation temperature, the thermophilic protein starts to denature The protein is an active hydrolase under single turnover conditions at room temperature
as demonstrated for stoichiometric loading with Mg-ATP by HPLC analysis (data not shown) Notably, the catalytic fragment is still active at a temperature of
30C, which is important with regard to our approach
to investigate the molecular reaction mechanism using time-resolved FTIR spectroscopy (see below)
Table 1 Binding of nucleotides to the catalytic fragments of
CPX-ATPase CopB The interaction is quantified from apparent binding
constants obtained by competitive binding titration of mant-ATP in
the presence of various concentrations of nucleotides Unless
indicated otherwise, Mg 2+ was omitted to prevent phosphatase
activity.
Nucleotide Binding constant Kdisslig (l M ) a
mant-ATP b ⁄ 1 m M MgCl2 10.0
a According to Eqn (4) b For mant-ATP in the absence of
competi-tor, the value for Kdissis given.
Trang 5Molecular interaction of ATP with CopB-B
Transient reactions were routinely observed using
rapid mixing techniques However, these are difficult
to perform in the case of time-resolved FTIR
spectros-copy The use of cuvettes with an optical path length
of less than 10 lm is imperative due to the high
absor-bance of water in the infrared region Under these
cir-cumstances, the reaction mechanism of the ATPase
can best be studied by release of ATP from the caged
precursor compound cgATP by photochemical
activa-tion according to the following reacactiva-tion scheme:
where kph represents the kinetic constant describing the fast photolytic cleavage of the caged compound It
is clear from equilibrium binding of cgATP (Table 1) that the CopB-BÆcgATP complex has already formed before photolysis To this end, samples were prepared
in special FTIR cuvettes with high concentrations of CopB-B and the Mg2+ complex of cgATP The com-ponents were present at a 1 : 1 ratio in order to pre-vent more than a single catalytic turnover Upon light activation for an integrated duration of 0.12 s, the genuine substrate is released
In order to clearly differentiate the post-flash IR absorbance signals into the photochemical processes of ATP release [25] and the subsequent hydrolytic protein reactions, the photochemical non-enzymatic process, which is strongly dependent on temperature and the
pH of the medium, must be the fastest reaction step The rapid appearance of positive absorbance changes
at 1123 cm)1 generated from free cgATP (Fig 4A, continuous line) and from cgATP in the presence of CopB-B (Fig 4A, dotted line) within the phosphate region of the infrared spectrum is indicative of product formation This band was assigned to the symmetric stretching vibration of the c-PO3 )group of ATP [25], thus providing information on the photochemical release rate of ATP from its caged precursor molecule The time course of the difference band corresponds to rates of 4 and 7 s)1 in the presence or absence of CopB-B, respectively, which demonstrates that the release of ATP is much faster than all subsequent partial reactions (see below), and, furthermore, gives a constant reference line for the pre-photolytic state of CopB-BÆATP after less than 2 s (Fig 4A)
Static photolysis spectrum and phosphate band assignment
The absorbance difference bands that are directly visi-ble in the spectra after photolysis of cgATP and those resolved by global fit analysis (see below) were assigned using substrate isotopologues [26] The IR difference spectrum recorded directly after photo-release indicates the binding state of the pre-existing CopB-BÆATP com-plex before the start of hydrolysis (Fig 4B) Negative difference bands at 1525 and 1347 cm)1 refer to the symmetric and anti-symmetric vibrations of the NO2 group in cgATP identified previously [25] For compari-son and further band assignment, spectra were run under identical conditions with ATP isotopically labelled at specific positions, i.e by chemical substitu-tion of 16O for 18O in the phosphate groups The increase in weight results in higher reduced masses of the molecular oscillators and therefore lowering of the
A
B
Fig 3 Catalytic properties of CopB-B (A) Substrate kinetics of
10 l M CopB-B with Mg-ATP at 70 C (B) Temperature dependence
of 10 l M CopB-B at an Mg-ATP concentration of 5 m M The pH of
the Na ⁄ Mes incubation medium at various temperatures was kept
constant between 5.9 and 6.2.
Scheme 1.
Trang 6vibrational frequencies As a typical example, Fig 4C
shows the photolysis spectrum of CopB-B with ATP
and c-18O4-ATP, respectively The positive band at
1137 cm)1 observed in the 16O compound is
down-shifted to 1089 cm)1 in the c-18O4-labelled ATP, and
this band can therefore be assigned to the
anti-symmet-ric stretching vibration of the c-phosphate group [ma (c-PO3 ))] Minor deviations of the observed band frequencies from tabulated values could relate to the
pH dependence of phosphate resonances and their shifts induced by formation of Mg complexes [25,27] Further band assignments are summarized in Table 2 (corresponding spectra not shown) It is worth noting that, in the CopB-B-bound state, the phosphate vibrations are coupled, as seen for example in the absorbance band at 1123 cm)1, which is shifted to
1101 cm)1 irrespective of placement of the18O label in the b or a group Strong phosphate coupling is other-wise known only for nucleotides in free aqueous solu-tion [26] In sharp contrast to CopB-B, phosphate coupling is abolished in the case of the GTP-binding protein Ras, in which phosphate absorbances are significantly shifted with respect to the non-bound state [26] and coupling between the a and b groups is removed The close similarity of IR difference spectra
of nucleotides in the presence and absence of CopB-B leads to the conclusion that the phosphate groups of ATP apparently do not contribute significantly to the formation of the nucleotide–protein complex; instead they are positioned in a hydrophilic environment or even remain solvent-exposed
Dynamic interaction of ATP with CopB-B:
time-resolved hydrolysis spectra revealing a reaction intermediate
After rapid release of the substrate ATP, its hydrolysis was observed to occur at comparatively low rates As
a control, the time course of the absorbance changes after photo-release was recorded in the spectral range from 1000–1800 cm)1 in the absence of protein, which demonstrates insignificant spectral contributions from cgATP and its photolysis alone (for details, see Fig S2) Upon elimination of the data related to the
A
B
C
Fig 4 Investigation of the ATPase reaction by FTIR spectroscopy (A) Time course of ATP photo-release from cgATP The absorbance changes of the symmetric coupled a,b-phosphate band of ATP at
1123 cm)1 (cgATP photolysed in presence of CopB-B, continuous line; cgATP photolysed alone, dotted line) were recorded by rapid-scan FTIR spectroscopy (B) Photolysis spectra of cgATP in the presence (continuous line) and absence of CopB-B (dotted line) The difference spectrum was obtained after 2 s, when ATP was fully released (C) Principle of band assignment of phosphate absor-bance difference bands in the photolysis spectrum by means of
18 O-labelled phosphates (dotted line) The reference spectrum (continuous line) was obtained with unlabelled ATP The absor-bance difference band (hatched upwards) is downshifted to another position (hatched downwards) in the spectrum obtained using c-18O 4 -labelled ATP under otherwise identical conditions.
Trang 7extremely fast initial photolytic phase (Fig 4A), the
relatively slow hydrolytic reaction rates were
kinetical-ly anakinetical-lysed by global fitting We were able to simulate
the spectral absorbance changes by multi-exponential
regression analysis with two rate constants k1app and
k2app Thus, to describe the overall hydrolysis reaction,
we derived a tentative working model displayed in
Scheme 2, consisting of the pre-hydrolytic initial state
(CopB-BÆATP), an intermediate (I) and a final state
(CopB-BÆADP):
In addition to the quickly formed so-called
photoly-sis spectrum ‘CopB-BÆATP–CopB-BÆcgATP’ (Fig 4B),
the consecutive reaction of the three protein states
connected by the two apparent rate constants is
repre-sented by two amplitude difference spectra )a1 and
)a2for the two rate constants k1app(Fig 5A, top) and
k2app (Fig 5A, bottom) Under the applied reaction
conditions, the first amplitude spectrum (k1app) could
be resolved with a rate constant of 1.9· 10)2s)1
(Fig 5A, top) and the second with a rate constant
(k2app) of 5· 10)3s)1(Fig 5A, bottom)
Kinetic modelling of CopB-B’s ATPase reaction
If the apparent rate constants k1app and k2app derived
from the global fitting differ only by a factor of four,
as in our case (Table 3), analysis of the spectral com-ponents of the amplitude spectra )a1 and )a2
(Fig 5A) becomes complicated due to mixing of states
In such a case, apparent and intrinsic rate constants often deviate drastically from each other For deter-mination of intrinsic rate constants for the ATP hydro-lysis, we applied the kinetic modelling program KinTek Global Kinetic Explorer [28] using the fol-lowing model (Scheme 3) with intrinsic rate constants
k1, k)1, k2and k)2:
In order to determine the intrinsic rate constants, we assumed that the concentration changes of CopB-BÆATP, the intermediate I and Piare proportional to the absorption changes at 1255 cm)1 (vas a-b-ATP band),
1338 cm)1 (unidentified protein side chain band) and
1078 cm)1 (inorganic phosphate band), respectively In addition, we normalized both the starting reactant (educt) absorbance at 1255 cm)1and the product absor-bance at 1078 cm)1, so that c0 (CopB-BÆATP) = c¥ (Pi) = 1 and c¥(CopB-BÆATP) = c0 (Pi) = 0 Due to the unknown absorption coefficient of the intermediate
I, we arbitrarily averaged both normalization factors for CopB-BÆATP and Pito obtain a reference for its relative concentration Based on these assumptions, we consid-ered models 1 and 2 described below
Model 1 is a simulation based on free parameter optimisation of the program, and yields k1= 4.7·
10)3s)1, k)1= 3.0· 10)4s)1, k2= 1.7· 10)2s)1and
Table 2 Assignment of phosphate vibration detected in the Mg-adenine nucleotide complexes of CopB-B by means of 18 O-labelled ATP iso-topologues.
Spectrum according
to global fit v (cm)1) Band assignment
Band position after shift upon addition of isotopolog
Deflection of the difference band d
18 O4-c (cm)1) 18 O3-b (cm)1) 18 O2-a (cm)1)
a
Assignment to more than one phosphate group indicates strong vibrational coupling [27].bsp., superposed Absorbance difference bands disappear upon isotopic labelling, but shifts are not observed due to complex band superposition c Amplitude spectra corresponding to the apparent rate constants k1app and k2app due to global fitting d u = upward, d = downward.
Scheme 2.
Scheme 3.
Trang 8k)2=1.0· 10)4s)1(Table 3) The corresponding con-centration profiles of the three components (Fig 6A) agree well with our normalized data (squares), indicat-ing reasonable selection of scalindicat-ing factors The main fea-tures of this kinetic model are that k2> k1(k2 k1app;
k1 k2app), and that back reactions are negligible The faster decline of the intermediate compared to its forma-tion leads to only small concentraforma-tions of intermediate I during the reaction The maximum concentration of I is approximately one-eighth of that of c0 (CopB-BÆATP) This is similar to the relatively small absorbance change
at 1338 cm)1compared to 1078 or 1255 cm)1, and thus
in line with our measurements
In model 2, parameters were fixed as suggested by global fitting, namely k1> k2 and k1 = k1app, and
k2= k2app and k)1= k)2= 0 Given these assump-tions, Fig 6B shows that the measured normalized absorbance at 1255 cm)1, indicative of the time course
of educt concentration, clearly deviates from its calcu-lated concentration profile Moreover, this simulation yields notably higher concentrations of the intermedi-ate than the former model
To further check the rationality and stability of our model assumptions, we varied the extinction coefficient
of the intermediate I for both models 1 and 2 (see Dis-cussion and Fig S3) In neither case did the simulated curves give better fits to the measured data than the ones displayed in Fig 6A Of even greater significance than the extinction coefficient of the intermediate I are the concentration profiles of educt and product, which both match optimally with curve fit 1 In summary, fit
1, based on program-chosen intrinsic constants, maps the time course of the reactant concentrations much better than fit 2, based on fixed constants; fit 1 therefore supports a credible model The data from model 1 were thus used to calculate the relative contributions of the states to the amplitude spectra )a1 and )a2 of the global fit as detailed in Appendix S1 The result of this calculation is that the bands facing upwards in )a1
(Fig 5A, top) derive from the intermediate state, and
A
B
Fig 5 FTIR spectroscopic measurement of the ATPase reaction as
performed by CopB-B, initiated by flash-initiated substrate liberation
of ATP from cgATP Rapid scan spectra recorded with a repetition
time of 185 ms (using double-sided forward–backward mode) fitted
to two rate constants by global fit analysis, k1app = 1.9 · 10)2s)1
and k 2app= 5 · 10)3s)1, starting from 2 s after the flash The band
labelled X is an artefact that also occurs in the sample without
pro-tein (A) Amplitude spectra corresponding to the rate k1app ( )a 1 , top)
and the rate k 2app ( )a 2 , bottom) (B) Band assignment verifying
phosphate production in the k 2apptransition by comparison of
ampli-tude spectra recorded with 16 O (continuous line) and 18 O (dotted
line) ATP isotopologues (top) and after double difference calculation
(16O–18O difference spectra) (bottom) The hatched zones indicate
the loss of c-ATP in the precursor state and the formation of
inorganic phosphate at the final stage of the phosphatase reaction.
Table 3 Kinetic constants obtained by various theoretical methods
of examination.
Kinetic stepa Rate constantb(s)1)
a
The steps are defined according to Schemes 2 or 3.bRate con-stants were calculated by data approximation via global fit [apparent rate constants (kiapp )] or via kinetic modelling (model 1; ki).
Trang 9the bands facing downwards derive from the final ADP
state The intensities are 38% compared to pure states
The bands facing downwards in )a2 derive from both
the intermediate state (38%) and the initial ATP state,
and the bands facing upwards in )a2 derive from the
final ADP state
The intermediate state during ATP hydrolysis
As the absorbances of the intermediate are facing
upwards in )a1 and downwards in )a2 (Fig 5A), the
appearance and disappearance of a band at 1338 cm)1 may be regarded as a marker of this intermediate It represents an unknown absorbing group of the protein, because absorbances in this region are clearly distinct from the phosphate vibrations Furthermore, the amplitude spectra displayed in Fig 5A indicate signifi-cant changes in the broad amide I band centered at approximately 1650 cm)1, and especially pronounced
at 1676 cm)1, and in the amide II band position at
1546 cm)1 This is not unexpected, as it is known that P-type ATPases undergo remarkable structural changes during catalysis Another interesting feature is the reproducible occurrence of small positive and nega-tive absorbance difference signals in the carbonyl region of the IR spectra in the region of 1720–
1740 cm)1, seen in both the )a1 and )a2 amplitude spectra (Fig 5A) Signals in this region point to the prevalence of protonated aspartic or glutamic acid side chains either undergoing protonation⁄ deprotonation reactions or conformational reorganizations
End product state of CopB-B-catalysed ATP hydrolysis
As mentioned above, the bands of the end product are the bands facing upwards in )a2 (Fig 5A, bottom) The shift of the positive band from 1078 to 1043 cm)1 upon c-18O-ATP labelling clearly demonstrates the for-mation of free inorganic phosphate in the product state, which becomes obvious in the absorbance differ-ence, and especially in the double difference spectrum (Fig 5B) Further product bands are found at 1220 and 1098 cm)1, which are assigned to the a and b vibrations of the hydrolysis product ADP (Table 2) Isotopic labelling at the c-18O-ATP position shifts the negative ms c-ATP band from 1136 to 1108 cm)1 (Fig 5B, curved arrow) As expected, the negative bands at 1255 and 1136 cm)1 (Fig 5A, bottom) corre-spond well with the positive bands in the photolysis spectrum (Fig 4B) from a-, b- and c-coupled ATP vibrations (Table 2)
Discussion
CopB-B is a suitable model to study ATP hydrolysis of the P-type ATPase CopB
We have measured significant basal ATPase activity of CopB in absence of the heavy metals (M Zoltner &
M Lu¨bben, unpublished observations) Similarly, metal-independent hydrolytic activity has also been observed with the CPX-ATPase CopA of Thermo-toga maritima [29] CopB-B can mimic the effects of
A
B
Fig 6 Time course of computed reactant concentrations after
kinetic modelling of the reaction between CopB-B and ATP The
normalized concentrations of reactants were plotted as fractions of
1 over time (educt CopB-BÆATP, red line; reaction intermediate I,
black line; product inorganic phosphate Pi, blue line) In addition,
the normalized measured absorbances of educt at 1255 cm)1
(CopB-BÆATP), of reaction intermediate at 1338 cm)1(unidentified
protein functional group) and of product at 1078 cm)1 (inorganic
phosphate Pi) are plotted (squares) Simulations were performed
under the two conditions: fit 1, for which intrinsic rate constants
k1, k2, k)1and k)2were optimized using the program KinTek Global
Kinetic Explorer (continuous lines) (A), and fit 2, for which fixed
rate constants k 1 = k 1appand k 2 = k 2app, k)1= k)2= 0 were chosen
(B).
Trang 10CopB-ATPase, which are entirely independent of the
translocated heavy metals, as the fragment naturally
carries out ‘uncoupled’ hydrolytic activity Our efforts
demonstrate that spectroscopic methods can be used to
study the substrate binding and catalytic activity of the
hyperthermophilic Sulfolobus enzyme CopB, because it
is easily handled at room temperature The catalytic
fragment CopB-B, consisting of nucleotide-binding
and phosphorylation domains, is the natively folded
‘business end’ of the holoenzyme CopB It is expected
that this fragment, whose 3D structure is known,
behaves similarly to the holoenzyme with respect to
ATP hydrolysis and thus serves as a model of it The
protein is capable of forming an intermediate with
covalently bound inorganic phosphate [24], and has
considerable ATPase activity despite the absence of the
actuator domain (A domain), which is considered to
promote rapid cleavage of the aspartyl phosphate
bond in Ca-ATPase [30] At 30C, the ATP hydrolysis
rate of CopB-B is fairly low, but still allows
observa-tion of the reacobserva-tion with substrate produced from
cgATP under single turnover conditions with a half-life
of approximately 3 min
Nucleotide binding to CopB-B
In order to precisely define the reaction conditions of
the spectroscopically observed CopB-B reaction with
ATP, the interaction of nucleotides with CopB-B was
explored by direct equilibrium binding or competition
assays using the fluorescent nucleotide mant-ATP As
has also been observed with other purine nucleotides,
cgATP has high affinity for CopB-B, which proves
that, within the applied concentration range of the
FTIR experiments ([cgATP]0>> Kdisslig (cgATP)), a
complex between the components has already formed
before photolysis After laser flash photolysis of
cgATP, the substrate ATP is released at the position
of its binding site, so this aspect of complex
associa-tion can be ignored for the kinetic interpretaassocia-tion of
our data
The nucleotide binding spectrum of CopB-B
obtained immediately after photolysis (Fig 4B) shows
a striking similarity to the spectrum of free ATP,
which is in sharp contrast to observations made with
several GTP-binding proteins such as Ras, Ran, Rab,
Rap and Rho, which exhibit vibrational uncoupling of
the phosphate resonances and significant shifts of the
a, b and c absorbance bands, resulting from strong
interactions of phosphate groups with amino acid side
chains lining the nucleotide binding site of the protein
[26,31–34] It is concluded that, in CopB-B, the
phos-phates stay in contact with the solvent, and the tightly
bound ATP becomes immobilized by other molecular parts of the nucleotide, presumably the purine moiety, which apparently protrudes into a binding pocket formed by CopB-B as seen in Fig 1
CopB-B interacts with ATP in a multi-step process
ATP hydrolysis of CopB-B apparently includes two phases These are kinetically resolved by global fit analysis and reflect the formation and decay of a single observable reaction intermediate Given the many intermediates that have been recognized during the reaction mechanism of P-type ATPases [2,35], more than one intermediary state would also be expected to occur during observation of hydrolysis with FTIR spectroscopy For example, there is spectral evidence for protonated carboxyl groups, of which one is expected as a potential phosphate acceptor in P-type ATPases [13], within the absorbance region of 1720–
1740 cm)1 (Fig 5A,B) Spectroscopic signatures of a transiently phosphorylated aspartic acid, as demon-strated earlier for Ca-ATPase [12], could not be resolved in our samples Details on the as yet unre-solved catalytic steps may be disclosed after careful adjustment of reaction conditions by either freezing otherwise invisible intermediates or investigating site-specific mutants
Kinetic process of ATP hydrolysis Kinetic modelling requires theoretical values for cata-lytic events as an input, but delivers a more detailed interpretation of measured data than global fitting Obvious deviations from recorded absorbance data occur, as in fit 2 (Fig 6B), in which the intrinsic rate constants were arbitrarily chosen as equal to the apparent constants In contrast, concentration profiles closely matched the absorbance time courses in the case where the intrinsic constants were adjusted (fit 1, Fig 6A) The educt decrease (CopBÆATP) takes place with the slower intrinsic rate k1, and the product increase (Pi) proceeds with the faster rate constant k2 Therefore, a relatively low concentration of intermedi-ate is seen, as the decay rintermedi-ate k2 of intermediate I is faster than its production rate k1 The slower rate k1 should be associated to the first process after release of ATP, i.e the conformational change of CopB-B leading to the ‘closed conformation’ In this step, the hydrophilic environment of the phosphate groups of ATP is substituted by a specific catalytic environment within a binding pocket of the protein This should induce dramatic absorption changes within the