Spectroscopic investigation of the reaction mechanism of CopB-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 (K diss 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 30 °C 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 (k 1 = 4.7 · 10 )3 s )1 at 30 °C) 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 (k 2 =17· 10 )3 s )1 at 30 °C), 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. 6172 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS intermediate. 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- cytogenes and 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). C. Vo ¨ llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6173 the 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 K diss ) 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 K diss , the conditions were adjusted such that the concentrations of mant-ATP and protein were of the same order as the expected K diss . 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 mM 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; [E t ] = 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 ([L 0 ]) of ATP (squares), ADP (circles) and AMP (trian- gles) for determination of the apparent K app diss . 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. Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo ¨ llmecke et al. 6174 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS curve 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 K diss increasing to 40 lm at 100 mm NaCl or (NH 4 ) 2 SO 4 . Notably, binding does not require Mg 2+ ; the affinity is reduced by a factor of 10 in the presence of 1 mm MgCl 2 (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 K app diss 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 [L 0 ], the binding constant of the nucleotide can be determined from the slope of the linear plot of the apparent binding constants K app diss and [L 0 ]. With the ligand ATP, a binding constant K lig diss 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 K lig diss 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 K M of 1 mm, which reflects relatively poor kinetic substrate affinity compared with the thermodynamic ligand association constant K lig diss 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 Mg 2+ , which indicates that Mg-ATP is the substrate of CopB-B. Furthermore, the ATPase activity increased in the temperature interval between 20–70 °C (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 30 °C, 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 K lig diss (lM) a mant-ATP b 0.8 mant-ATP b ⁄ 1mM MgCl 2 10.0 ATP 10.0 ADP 18.9 AMP 29.8 AMPPNP 3.5 cgATP 9.5 GTP 12.6 a According to Eqn (4). b For mant-ATP in the absence of competi- tor, the value for K diss is given. C. Vo ¨ llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6175 Molecular 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 reaction scheme: where k ph 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 Mg 2+ 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-PO 3 2) 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 NO 2 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 16 O for 18 O 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 mM. The pH of the Na ⁄ Mes incubation medium at various temperatures was kept constant between 5.9 and 6.2. Scheme 1. Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo ¨ llmecke et al. 6176 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS vibrational frequencies. As a typical example, Fig. 4C shows the photolysis spectrum of CopB-B with ATP and c- 18 O 4 -ATP, respectively. The positive band at 1137 cm )1 observed in the 16 O compound is down- shifted to 1089 cm )1 in the c- 18 O 4 -labelled ATP, and this band can therefore be assigned to the anti-symmet- ric stretching vibration of the c-phosphate group [m a (c-PO 3 2) )]. 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 the 18 O 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- 18 O 4 -labelled ATP under otherwise identical conditions. C. Vo ¨ llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6177 extremely fast initial photolytic phase (Fig. 4A), the relatively slow hydrolytic reaction rates were kinetical- ly analysed by global fitting. We were able to simulate the spectral absorbance changes by multi-exponential regression analysis with two rate constants k 1 app and k 2 app . 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 )a 1 and )a 2 for the two rate constants k 1 app (Fig. 5A, top) and k 2 app (Fig. 5A, bottom). Under the applied reaction conditions, the first amplitude spectrum (k 1 app ) could be resolved with a rate constant of 1.9 · 10 )2 s )1 (Fig. 5A, top) and the second with a rate constant (k 2 app )of5· 10 )3 s )1 (Fig. 5A, bottom). Kinetic modelling of CopB-B’s ATPase reaction If the apparent rate constants k 1 app and k 2 app 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 )a 1 and )a 2 (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 ExplorerÔ [28] using the fol- lowing model (Scheme 3) with intrinsic rate constants k 1 , k )1 , k 2 and k )2 : In order to determine the intrinsic rate constants, we assumed that the concentration changes of CopB- BÆATP, the intermediate I and P i are proportional to the absorption changes at 1255 cm )1 (v as 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 )1 and the product absor- bance at 1078 cm )1 , so that c 0 (CopB-BÆATP) = c ¥ (P i ) = 1 and c ¥ (CopB-BÆATP) = c 0 (P i ) = 0. Due to the unknown absorption coefficient of the intermediate I, we arbitrarily averaged both normalization factors for CopB-BÆATP and P i to 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 k 1 = 4.7 · 10 )3 s )1 , k )1 = 3.0 · 10 )4 s )1 , k 2 = 1.7 · 10 )2 s )1 and 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 O 4 -c (cm )1 ) 18 O 3 -b (cm )1 ) 18 O 2 -a (cm )1 ) Photolysis 1123 v s a-b-ATP a 1101 1101 u 1137 v as c-ATP 1089 u 1213 v as b-a-ATP 1206 u 1250 v as a-b-ATP sp. b u )a 1 c 1108 ATP ⁄ ADP sp. d )a 2 1078 v s (PO 2 ) ) phosphate 1043 u 1098 v b-ADP sp. u 1136 v as c-ATP sp. d 1220 v as a-ADP sp. u 1255 v as a-b-ATP sp. sp. d a Assignment to more than one phosphate group indicates strong vibrational coupling [27]. b sp., 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 k 1 app and k 2 app due to global fitting. d u = upward, d = downward. Scheme 2. Scheme 3. Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo ¨ llmecke et al. 6178 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS k )2 =1.0 · 10 )4 s )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 scaling factors. The main fea- tures of this kinetic model are that k 2 > k 1 (k 2 $ k 1 app ; k 1 $ k 2 app ), and that back reactions are negligible. The faster decline of the intermediate compared to its forma- tion leads to only small concentrations of intermediate I during the reaction. The maximum concentration of I is approximately one-eighth of that of c 0 (CopB-BÆATP). This is similar to the relatively small absorbance change at 1338 cm )1 compared 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 k 1 > k 2 and k 1 = k 1 app , and k 2 = k 2 app 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 )a 1 and )a 2 of the global fit as detailed in Appendix S1. The result of this calculation is that the bands facing upwards in )a 1 (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, k 1 app = 1.9 · 10 )2 s )1 and k 2 app =5· 10 )3 s )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 k 1 app ()a 1 , top) and the rate k 2 app ()a 2 , bottom). (B) Band assignment verifying phosphate production in the k 2 app transition 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 ( 16 O– 18 O 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 step a Rate constant b (s )1 ) First k 1 app 1.9 · 10 )2 k 1 4.7 · 10 )3 k )1 3.0 · 10 )4 Second k 2 app 5.0 · 10 )3 k 2 1.7 · 10 )2 k )2 1.0 · 10 )4 a The steps are defined according to Schemes 2 or 3. b Rate con- stants were calculated by data approximation via global fit [apparent rate constants (k i app )] or via kinetic modelling (model 1; k i ). C. Vo ¨ llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6179 the bands facing downwards derive from the final ADP state. The intensities are 38% compared to pure states. The bands facing downwards in )a 2 derive from both the intermediate state (38%) and the initial ATP state, and the bands facing upwards in )a 2 derive from the final ADP state. The intermediate state during ATP hydrolysis As the absorbances of the intermediate are facing upwards in )a 1 and downwards in )a 2 (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 )a 1 and )a 2 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 )a 2 (Fig. 5A, bottom). The shift of the positive band from 1078 to 1043 cm )1 upon c- 18 O-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- 18 O-ATP position shifts the negative m s 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 P i , 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 P i ) are plotted (squares). Simulations were performed under the two conditions: fit 1, for which intrinsic rate constants k 1 , k 2 , k )1 and k )2 were optimized using the program KinTek Global Kinetic ExplorerÔ (continuous lines) (A), and fit 2, for which fixed rate constants k 1 = k 1 app and k 2 = k 2 app , k )1 = k )2 = 0 were chosen (B). Hydrolytic mechanism of the catalytic CPx-ATPase domain C. Vo ¨ llmecke et al. 6180 FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS CopB-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 30 °C, the ATP hydrolysis rate of CopB-B is fairly low, but still allows observa- tion of the reaction 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 >> K diss lig (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 interpretation 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 k 1 , and the product increase (P i ) proceeds with the faster rate constant k 2 . Therefore, a relatively low concentration of intermedi- ate is seen, as the decay rate k 2 of intermediate I is faster than its production rate k 1 . The slower rate k 1 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 phos- C. Vo ¨ llmecke et al. Hydrolytic mechanism of the catalytic CPx-ATPase domain FEBS Journal 276 (2009) 6172–6186 ª 2009 The Authors Journal compilation ª 2009 FEBS 6181 [...]... vast excess, offering an explanation of why the KM value of the ATPase reaction is relatively high compared with the lig fairly low equilibrium binding constant Kdiss of ATP Conclusions Partial reactions of the CPX-ATPase holoenzyme CopB can be investigated using the catalytic fragment CopB-B Despite the fact that no information on the fate of the translocated heavy metal ion can be obtained, the nucleotide-binding... concentration [L0], the apparent binding constant for mant-ATP is expressed as ! ẵ L0 app 4ị Kdiss ẳ Kdiss 1 ỵ lig Kdiss in which Kdiss represents the binding constant of the mant-ATP complex in the absence of competitor, and lig Kdiss represents the binding constant of the competitor lig ligand Kdiss may be read from the slope of the linear plot app of the apparent binding constant Kdiss versus the total concentrations...Hydrolytic mechanism of the catalytic CPx-ATPase domain phate region [26] The absence of more intense phosphate absorbance difference bands in our measurements further supports the kinetic model in which k2 > k1, because the concentration of the reaction intermediate is low, giving rise to only weak absorption changes Thus, we conclude that the rate-determining step of our reaction is the slow snapping... scans), 60600 s (4170; 100 scans) and 6001000 s (7172; 1000 scans) The averaged interferograms were manipulated by zero lling using a factor of 2, and Fourier-transformed using Mertz phase correction and the BlackmanHarris three-term apodization function Absorbance spectra and absorbance time courses are displayed as differences between the light intensity I(t) and the reference intensity I0 of the. .. snapping process of the domains to the intermediate I form with rate constant k1 The conformational rearrangements involved in this snapping are shown by the relatively large change in the amide I band upon intermediate formation, and the subsequent reversal of this change during the product formation Once this catalytically active conformation is formed, the subsequent processes are fast The reaction intermediate... performed as described above for the second method, but with the additional presence of 0, 5, 10, 20 or 25 lm of the competitor nucleotide In this case, the binding constant Kdiss of mant-ATP (in the absence of competitor) app increases to the apparent binding constant Kdiss (in the pres- ence of competitor) Under the assumption that the free concentration of competitor ligand [L] is negligible compared... Acknowledgements 15 We thank Dr Yan Suveyzdis for chemical synthesis of the isotopologues of cgATP and Ingo Rekittke for the preparation of mant nucleotides This work was supported by grants LU405 3-1 from the Deutsche Forschungsgemeinschaft and I 78128 from the VolkswagenStiftung to M.L 16 17 References 1 Palmgren MG & Axelsen KB (1998) Evolution of P-type ATPases Biochim Biophys Acta 1365, 3745 2 Kaplan JH (2002)... Biochemistry of Na,K-ATPase Annu Rev Biochem 71, 511535 3 Jứrgensen PL, Hakansson KO & Karlish SJ (2003) Structure and mechanism of Na,K-ATPase: functional sites and their interactions Annu Rev Physiol 65, 817849 4 Kuhlbrandt W (2004) Biology, structure and mechanism ă of P-type ATPases Nat Rev Mol Cell Biol 5, 282295 5 Toyoshima C & Inesi G (2004) Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic... 50 lm of CopB-B in small intervals to a constant concentration of mant-ATP (25 lm) (for details, see Results and Discussion) Data were obtained 30 s after addition of the ligand Read-outs were corrected for dilution due to the added volumes Titrations for determination of Kdiss were performed in two ways In the rst method, the concentration of mant-ATP or mant-ADP (0.5 lm) was kept constant, and small... saturating concentration of protein [At] represents the total concentration of ligand (independent variable), and [EA] is the actual concentration of the mant-ATPCopB-B complex, which is given by EA ẳ ẵEt ỵ Kdiss ỵ ẵAt ị ẵEt ỵ Kdiss ỵ ẵAt ị2 Synthesis of nucleotide analogues mant-ATP (see Fig 2A for structural formula) was synthesized using the procedure described previously [37] Synthesis of cgATP (see Fig . Spectroscopic investigation of the reaction mechanism of CopB-B, the catalytic fragment from an archaeal thermophilic ATP-driven heavy metal transporter Christian. 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