Tài liệu Báo cáo Y học: Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenol pyruvate carboxykinase determined by fluorescence spectroscopy pdf

Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phospho enol pyruvate carboxykinase determined by fluorescence spectroscopy Marı ´ a Victoria Encinas 1 , Fernando D. Gonza ´ lez-Nilo 1 , Hughes Goldie 2 and Emilio Cardemil 1 1 Departamento de Ciencias Quı ´ micas, Facultad de Quı ´ mica y Biologı ´ a, Universidad de Santiago de Chile, Chile; 2 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the c-phosphoryl group of ATP to yield PEP, ADP, and CO 2 . The interaction of the enzyme with the substrates ori- ginates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phos- phopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several pro- tein–ligand dissociation constants, as well as the role of Mg 2+ and Mn 2+ in substrate binding. The binding affinity of PEP to the enzyme–Mn 2+ complex was )8.9 kcalÆmol )1 , which is 3.2 kcalÆmol )1 more favorable than in the complex with Mg 2+ . For the substrate nucleotide–metal complexes, similar binding affinities ()6.0 to )6.2 kcalÆmol )1 )were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme–PEP–Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl–E. coli PEP carb- oxykinase showed different degrees of solvent-exposed sur- faces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution. Keywords: Escherichia coli phosphoenolpyruvate carboxy- kinase; ligand binding; conformational changes; P-pyridoxyl fluorescence spectroscopy. Escherichia coli phosphoenolpyruvate carboxykinase [PEP carboxykinase; ATP:oxaloacetate carboxylase (trans-phos- phorylating) EC 4.1.1.49] catalyzes the reversible decarb- oxylation of oxaloacetic acid (OAA) with the associated transfer of the c-phosphoryl group of ATP to yield PEP and ADP, where M 2+ is a divalent metal ion: OAA þ ATP ! M 2þ PEP þ ADP þ CO 2 The physiological role of this enzyme in bacteria and most other organisms is to catalyze the formation of PEP in the first committed step of gluconeogenesis [1]. The crystal structure of free- and substrate-bound E. coli PEP carb- oxykinase has been solved at 1.9 A ˚ resolution [2,3]. The enzyme is a monomeric, globular protein that belongs to the a/b protein class. The overall structure has two domains, a 275 residue N-terminal domain, and a more compact 265 residue C-terminal domain, with the active site in a deep cleft between them. The recently reported crystal structure of Trypanosoma cruzi PEP-carboxykinase [4] shows remarkable similarity. Upon substrate binding, the E. coli enzyme undergoes a domain closure through a 20° rotation of the two domains towards each other, excluding bulk solvent from the active site and positioning active site residues for catalysis [3]. Results obtained with AlF 3 complexes of E. coli PEP carboxykinase indicate that phosphoryl transfer occurs via a direct displacement mech- anism with associative qualities [5]. In spite of the detailed knowledge of the structural characteristics of E. coli carb- oxykinase, very little information is available for ATP- dependent carboxykinases with respect to thermodynamic data on ligand binding [6]. Chemical modification studies have shown that PLP specifically labels the protein in a lysyl residue located at position 288 and, upon reduction of the labeled enzyme with sodium borohydride, a P-pyridoxyl group is covalently attached at this site [7]. The crystal coordinates of the E. coli enzyme indicate that this residue, located in the C-terminal domain, is 9.7 A ˚ from Gly251, which is the closest amino acid residue of the N-terminal domain, in the P-loop of the enzyme. Upon domain closure, the distance from Lys288 to Gly251 reduces to 5.3 A ˚ , thus making Lys288 an excellent observation point to follow the domain movement of the protein in solution, provided this motion can be detected. Spectroscopic properties of the Schiff base formed upon reaction of PLP with amino acids or amines are highly dependent on medium properties such as pH or polarity [8,9]. Spectroscopic studies have been employed to obtain information about the mechanism of some PLP-dependent enzymes [10]. Reduction of the imine bond with NaBH 4 Correspondence to M. V. Encinas, Departamento de Ciencias Quı ´ micas, Facultad de Quı ´ mica y Biologı ´ a, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile. Fax: + 56 2 681 2108, Tel.: + 56 2 681 2575; E-mail: mencinas@lauca.usach.cl Abbreviations: OAA, oxaloacetic acid; PEP, phosphoenolpyruvate; PLP, pyridoxal 5¢-phosphate; P-pyridoxyl, phosphopyridoxyl. (Received 16 May 2002, revised 26 July 2002, accepted 21 August 2002) Eur. J. Biochem. 269, 4960–4968 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03196.x attaches the probe covalently to the protein, thus providing a suitable probe to detect structural conformational chan- ges, as its spectroscopic features are also expected to be highly dependent on the medium properties. In this work we took advantage of the specific labeling with PLP of Lys288 in E. coli PEP carboxykinase to gain insight into conformational alterations of the protein upon ligand binding in solution. Analysis of the fluorescent characteristics of the reduced Schiff base allowed us to obtain binding constants for the interaction of several substrates and ligands, and the role of metal ions on their binding. EXPERIMENTAL PROCEDURES Materials PLP, NaBH 4 , NaCNBH 3 , PEP, nucleotides, MnCl 2 , MgCl 2 , and pyridoxamine were from Sigma Chemical Co., OAA from Boehringher Mannheim, oxalate from Merck. Recombinant E. coli PEP carboxykinase was obtained as described [11]. All other reagents were of the purest commercially available grade. Labeling of E. coli PEP carboxykinase with PLP The enzyme (25–30 l M ) was reacted with a fourfold molar excess of PLP for 5 min at 0 °Cin50m M Hepes (pH 7.5) containing 3 m M NaCNBH 3 . The reaction was stopped with 100 m M NaBH 4 , and excess reagents eliminated by dialysis at 4 °C against 50 m M Hepes (pH 7.0). Under these conditions, PLP specifically reacts with Lys288 [7]. Labeling stoichiometries, determined from e 280 ¼ 67 700 M )1 Æcm )1 for E. coli PEP carboxykinase [2,12] and e 325 ¼ 9710 M )1 Æcm )1 for the P-pyridoxyl group [8], were in the range 0.8–1.0 mol P-pyridoxyl/mol of protein. Fluorescent measurements All measurements were carried out at 22 °Cin50 m M Hepes buffer (pH 7.0). Steady state fluorescence measurements were performed on a Spex Fluorolog spectrofluorometer with excitation and emission band width of 1.25 nm. The excitation wavelength to follow the tryptophan fluorescence was 295 nm, while 325 nm were used for the P-pyridoxyl fluorescence. All spectra were recorded using the corrected mode. Fluorescence lifetimes were measured with an Edinburgh Instruments OB 900 time correlated single photon counting fluorimeter, using a hydrogen filled lamp for excitation. Fluorescence quenching experiments with acrylamide were carried out by monitoring the decrease in intensity at the emission maximum wavelength or the change in the fluorescence lifetimes. Successive aliquots of freshly prepared solutions (5.6 M )wereaddedtoacell containing the protein, and the respective parameter was measured. Appropriate corrections were made for dilution effects (never exceeding 10%). The quenching data were fitted to the Stern–Volmer equation, F  =F ¼ 1 þ K SV ½Qð1Þ where F ° and F are the fluorescence intensities in the absence and presence of quencher, respectively. K SV is the Stern–Volmer constant, which is related to the bimolecular quenching rate constant (k q ) and the lifetime of the singlet excited state in the absence of quencher (s°)byK SV ¼ k q Æs°. Values of k q were calcu- lated using the amplitude average lifetimes Æ sæ ¼ Sf i s i , where f i is the amplitude fraction. The effect of ligands was analyzed by monitoring the change of fluorescence intensity upon ligand addition to protein solutions. OAA solutions were prepared just before the experiments. The decomposition of OAA under our experimental conditions was determined using the lactic dehydrogenase assay [13], and was lower than 12%. Concentration of CO 2 is expressed as total bicarbonate. The dissociation equilibrium constants (K d )ofthe protein–ligand (LP) complexes were evaluated by the curve fitting to the quadratic equation deduced from the equilib- rium L + P , LP, considering that LP is proportional to the emission intensity changes. Y F ¼ðF obs À F o Þ=ðF 1 À F o Þ¼½ðP þ L þ K d Þ ÀððP þ L þ K d Þ 2 À 4P ðLÞ 1=2 =2P ð2Þ Where F obs is the measured fluorescence intensity, F o is the fluorescence intensity at the start of the titration, F ¥ is the fluorescence intensity at saturating concentration of ligand, P the total protein concentration, and L is referred to the ligand concentration. The distribution of metal ion as [M 2+ ] free , [ML] and of [L] free was calculated using the dissociation con- stants for the individual species present. The values used were MnÆoxalacetate ¼ 1.2 · 10 )2 M [14], MnÆoxa- late ¼ 1.78 · 10 )4 M , MgÆoxalate ¼ 4.17 · 10 )3 M [15] MnATP 2– ¼ 1.5 · 10 )5 M , MnHATP – ¼ 2.2 · 10 )3 M , MgATP 2– ¼ 6.3 · 10 )5 M , MgHATP – ¼ 4.8 · 10 )3 M , MnADP – ¼ 8.1 · 10 )5 M , MnHADP ¼ 1.3 · 10 )2 M , MnAMP ¼ 4.3 · 10 )3 M , MnPEP – ¼ 5.5 · 10 )3 M , MgPEP – ¼ 1.8 · 10 )3 M [16]. For MnGDP – and MnHGDP the values of the corresponding ADP complexes were used. The concentration of the different species at pH 7.0 was calculated using the program COMPLEX version 6 (1986) written by A. Cornish-Bowden, Centre National de la Recherche Scientifique, Marseille, France. Computer-assisted three-dimensional modeling The programs INSIGHTII and DISCOVER 972 (MSI) were used on an O2 SGI workstation to obtain the three-dimensional models of E. coli PEP carboxykinase. The structures analyzed were E. coli PEP carboxykinase (1OEN) [2] and the E. coli PEP carboxykinase–Mg 2+ -Mn 2+ –ATP–pyru- vate complex (1AQ2) [3]. Amino acids lost in crystallo- graphic data were inserted into each structure. All calculations were carried out with DISCOVER _3 (MSI) and force field CVFF and ESFF (MSI), that has all parameters needed for the octahedral Mn 2+ coordination and amino acids (1AQ2). This program was also employed for energy minimization and molecular dynamics. The metal ion Mg 2+ was replaced by Mn 2+ in octahedral coordination to three water molecules, a bidentate coordination to two oxygen atoms from P b and P c of ATP, and the oxygen of the hydroxyl group of Thr255. The second Mn 2+ was in octahedral coordination to two water molecules, an oxygen atom from P c of ATP, N e2 from His232, an oxygen atom from the side chain of Asp269, and N e from Lys213. Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 4961 N e from Lys288 of the open structure was covalently linked to carbonyl group of PLP through an imino linkage. Then, the best position of the P-pyridoxyl group was selected by performing an energy barrier calculation of the dihedral angle C-Ne-O-P [17], and the resulting structure was relaxed using a cycle of simulated annealing. Initially, the system was gradually heated from 200 to 500 K, with increments of 37.5 K for 5 ps each. Then, the system was equilibrated at 500 K by 10 ps, and finally cooled to 200 K, decreasing 10 K each 5 ps (30 steps). The relaxed structure obtained was finally minimized using steepest descents and conjugate gradients algorithms. This final position for the P-pyridoxyl group was used as a starting structure in the closed E. coli PEP carboxykinase model, and then it was minimized using a simulated annealing by 200 ps. The cutoffs for van der Waals and Coulombic interactions were 10 A ˚ and 12 A ˚ , respectively. Using both final structures, the solvent acces- sible surface area of the P-pyridoxyl group and of active site residues were calculated using a solvent radius of 1.4 A ˚ (water). For the amino acids, the fraction of solvent accessible surface area was calculated using the Gly-X-Gly tripeptide model implemented in INSIGHTII [18]. The residues on either side of the index residue are mutated to glycine, and the solvent accessible surface area for the index residue is calculated (reference value for 100% solvent accessible surface). The reference value for each residue is dependent on the conformation of the neighboring residues. RESULTS Characteristics of the P-pyridoxyl group linked to E. coli PEP carboxykinase Free pyridoxamine, which can be considered as a model of a pyridoxyl group bound to a Lys residue, exhibits an absorption spectrum with a maximum at 326 nm at pH 7 and 20 °C. This band can be assigned to the bipolar form of the pyridoxamine [8], as consequence of the deprotonation of the phenolic group. Upon excitation at 326 nm, pyridoxam- ine shows a well shaped emission band centered at 393 nm at pH 7. The absorption spectrum of the P-pyridoxyl adduct of PEP carboxykinase exhibits a band with a maximum at 326 nm due to pyridoxyl moiety and a band at 280 nm corresponding to the aromatic amino acids of the protein. The fluorescence of the P-pyridoxyl moiety bound to the protein at pH 7 is similar to that of free pyridoxamine, with a maximum at 393 nm. This spectral behavior reflects a high degree of exposure of the P-pyridoxyl group to the solvent. The fluorescence decay of pyridoxamine and of the P-pyridoxyl-labeled protein were monitored at 393 nm upon excitation at 326 nm. The emission decay of pyridox- amine was monoexponential with a lifetime of 1.83 ns, while that of P-pyridoxyl bound to the protein could only be fitted by two exponential decays of 5.15 ns and 1.21 ns, with fractional intensities of 0.34 and 0.66, respectively (Fig. 1). This heterogeneous emission decay indicates that the pyridoxyl chromophore senses microheterogeneous envi- ronments during its lifetime due to its localized motion, to relaxation processes involving the solvent, and/or adjacent residues on the protein surface. To get an approximation of the steric relationships between the Lys288-bound P-pyridoxyl group and the protein structure, the corresponding complex was modeled using the crystalline coordinates of the free E. coli PEP carboxykinase [2]. The deviation of the resulting model structure (P-pyridoxyl labeled protein) from the coordinates of the starting structure (PDB: 1OEN), give a r.m.s. value of 0.95 A ˚ for Ca. Figure 2 shows that the P-pyridoxyl group is located close to active site in a position that allows the access of substrates to the active site. When the amino acid residues located £ 4A ˚ from ATP and the two metal ions were considered, it was found that in the open, ligand-free structure, the P-pyridoxyl group does not overlap any residue except the Thr251, which corresponds to only 4.7% of the total solvent accessible area considered. The Thr251 is a noncatalytic residue close to C2¢ of ATP. Thus, this location makes this chromophore a suitable probe to sense conformational changes that occur in this protein region upon substrate binding. Effect of ligands on the P-pyridoxyl fluorescence The addition of metal ions or substrates to the labeled enzyme caused marked changes in the emission character- istics of the bound P-pyridoxyl group (Fig. 3). The addition of Mn 2+ quenched the fluorescence. However, the addi- tion of PEP or ATP in the presence of saturating concentrations of Mn 2+ increased the fluorescence intensity. These fluorescence variations suggest that Fig. 1. Fluorescence decay profiles of P-pyridoxyl bound to the E. coli PEP carboxykinase in Hepes pH 7.0, k exc ¼ 326 nm, k em ¼ 393 nm (a) in absence of substrates; (b) in the presence of 1 m M PEP plus 2 m M Mn 2+ (c) instrumental response function. The solid line corresponds to a biexponential function with s 1 ¼ 5.15 ns (34%), s 2 ¼ 1.21 ns for the enzyme–adduct in the absence of substrate, and s 1 ¼ 6.10 ns (51%), and s 2 ¼ 1.14 ns in the presence of PEP and Mn 2+ . Bottom: distri- bution of residuals. 4962 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 conformational changes caused by the ligand are easily sensed by the P-pyridoxyl chromophore. Figure 4 shows the modeled structure of the P-pyridoxyl-PEP carboxykinase in the free and substrate bound conformations. In the open conformation (Fig. 4A), the P-pyridoxyl moiety is rather solvent exposed with a fractional exposed area of 0.39, meanwhile in the closed conformation (Fig. 4B) the exposed area is 0.082, indicating that the fluorophore is now almost completely hindered into the protein matrix. The addition of Mn 2+ , an essential metal ion for catalysis, to the labeled PEP carboxykinase caused the quenching of the fluorescence signal without any shift of the spectrum. The pattern of fluorescence quenching by Mn 2+ was biphasic (Fig. 5). The first phase occurred approxi- mately in the range from 0 to 0.3 m M Mn 2+ , whereas the second phase implies a lower quenching that occurred at millimolar concentrations of the metal ion. This biphasic behavior indicates the presence of binding sites with different affinities. Data of fluorescence intensity as function of Mn 2+ concentration (Fig. 5) were well fitted to Eqn (2), expressed as a double binding function. Values of K d of 17.4 l M and 1.4 m M were obtained for the high and low affinity binding sites, respectively (Table 1). When similar experiments were carried out with Mg 2+ , fluorescence quenching was observed only at metal ion concentrations in the millimolar range, and the data were well fitted to a monophasic saturation curve with K d of 1.8 m M .These results imply a low affinity site for the magnesium cation in the protein. The incubation of the labeled enzyme with increasing concentrations of adenine nucleotides in the presence of Mn 2+ led to a progressive enhancement of the emission, and to a blue shift of approximately 4 nm in the emission maximum. The increase of the fluorescence intensity gave a monophasic saturation curve. Data obtained for different nucleotides are displayed in Fig. 6A. The dissociation constants, calculated assuming that nucleotides bind as nucleotide–metal complex [3,19], are given in Table 1. These values show similar affinities for MnATP and MnADP, and much lower affinity for the metal monophosphorylated nucleotide derivative (Table 1). This is in agreement with the expected requirement of the b-andc-phosphate groups of the nucleotide for efficient binding to the protein active site [20]. Binding affinity of MgATP was similar to that in the presence of Mn 2+ , and also similar to previous determina- tions of ATP and ADP binding to the native E. coli PEP carboxykinase [6]. On the other hand, the addition of ATP or ADP in the absence of metal ions gave emission changes at much higher nucleotide concentrations, suggesting binding to low affinity, noncatalytic sites. The addition of GDP in the presence of Mn 2+ to the labeled protein produced changes in the fluorescence emission only at high concentrations (Fig. 6A), as expected from the known specificity of the E. coli PEP-carboxykinase for adenosine nucleotides [20,21]. Thus, results obtained from nucleotide binding to the P-pyridoxyl-labeled enzyme, show that the enhanced emission in the presence of ADP or ATP can only be a consequence of conformational changes caused by the binding of the nucleotide to the enzyme active site region. Binding of CO 2 (expressed as total bicarbonate), another substrate of the enzyme, also increased the P-pyridoxyl fluorescence. The addition of this substrate to the protein blue shifted the maximum by 5 nm and the fluorescence Fig. 3. Steady state fluorescence spectra of 2 l M P-pyridoxyl-E. coli PEP carboxykinase using k exc ¼ 326 nm, in the presence of different combinations of substrates and metal ions: (a) in the absence of ligands; and in the presence of (b) 2 m M Mn 2+ (c) 1 m M ATP plus 2 m M Mn 2+ (d) 0.05 m M PEP plus 1 m M Mn 2+ . Fig. 2. Molecular model of the P-pyridoxyl-E. coli PEP carboxykinase adduct. N e from Lys288 of the open structure of E. coli PEP carb- oxykinase (1OEN) is covalently linked to the carbonyl carbon of the P-pyridoxyl group (PL) through an imino linkage. The green line shows the protein backbone, the P-pyridoxyl group is shown in yellow. The Connelly surface of the active site residues is shown in magenta. Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 4963 increased 1.52-fold. Interestingly, CO 2 binding was not affected by the presence of cations. The saturation curves in the absence or presence of Mn 2+ were similar, K d in the presence of Mn 2+ was 13.7 m M which is close to the apparent K m for HCO 3 – (13 m M ) [21]. Also, a similar dissociation constant of 8.2 m M has been determined for the enzyme–CO 2 complex of homologous Saccharomyces cere- visiae PEP carboxykinase [22]. The addition of PEP to the labeled protein in the absence of divalent cations produced no changes in the emission properties of the P-pyridoxyl chromophore. However, in the presence of saturating concentrations of Mn 2+ , micromolar concentrations of PEP produced notable changes on the P-pyridoxyl fluorescence (Fig. 6B). The intensity increased almost threefold and the emission maximum was blue shifted by 10 nm. The fitting of data to Eqn (2) using the free PEP concentration gave K d value of 0.25 l M (Table 1) . Fluorescence data using the MnPEP concentration did not fit to Eqn (2). Binding of PEP in the presence of Mg 2+ was also accompanied by the enhancement of the fluorescence intensity and a spectral shift of 7 nm. However, the dissociating constant was two orders of magnitude higher than that obtained in the presence of Mn 2+ (Table 1). An independent estimation of the binding affinity of PEP for E. coli PEP carboxykinase was obtained from the quenching of the emission of Trp residues of the unlabeled protein. When the protein was titrated with PEP in the presence of saturating concentrations of Mn 2+ , the intrinsic fluorescence was quenched by 10%. In spite of this small effect of PEP on the Trp emission, the fluorescence decrease as function of free PEP concentration gave a saturating plot thatfittedtoEqn(2)withaK d of 0.22 l M (Table 1). This value is similar to that obtained using the modified enzyme. This indicates that both types of signals, Trp and P-pyri- doxyl fluorescence, monitor the same process. Furthermore, this indicates that derivatizing PEP-carboxykinase with Fig. 4. Space-filling diagrams of the P-pyri- doxyl-E. coli PEP carboxykinase adduct in the open (A) and closed (B) structures. The N-ter- minal domains of the two structures are colored yellow, and the C-terminal domains green. The phosphoryl and pyridoxyl moieties of the P-pyridoxyl group are shown in red and magenta, respectively. The fractional solvent exposed area of the P-pyridoxyl group is 0.39 and 0.082 for the open and closed structures, respectively. The molecular models for (A) and (B) are based on PDB structures 1OEN and 1AQ2, respectively. Fig. 5. Changes in the fluorescence of P-pyridoxyl-E. coli PEP carb- oxykinase (3 l M )asfunctionofMn 2+ concentration. The solid line represents the fitting of data to Eqn (2) expressed as a double binding function. Table 1. Dissociation equilibrium constants for the ligand-protein com- plexes. Ligand Metal ion a K d (l M ) Mn 2+ – 17.4 ± 3.6 (40 ± 6) b 1400 ± 480 Mg 2+ – 1800 ± 380 ATPMn Mn 2+ 24 ± 1.0 ATPMg Mg 2+ 33.8 ± 2.0 (10 ± 1) b ADPMn Mn 2+ 24.8 ± 1.0 (18 ± 2) c AMPMn Mn 2+ 128 ± 7 GDPMn Mn 2+ 508 ± 37 PEP Mn 2+ 0.24 ± 0.02 (0.21 ± 0.08) c PEP Mg 2+ d 53.8 ± 4.4 CO 2 – 10 300 ± 350 CO 2 Mn 2+ 13 700 ± 600 Oxalacetate Mn 2+ 156 ± 17 Oxalate Mn 2+ 26 ± 2 Oxalate Mg 2+ d 136 ± 16 a Metal ion concentration was 2 m M in all cases. b From [6], cal- culated from the Trp fluorescence quenching in the unlabeled enzyme. c This work, calculated from the Trp fluorescence quenching in the unlabeled enzyme. d Mg 2+ ,4m M . 4964 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 P-pyridoxyl group does not alter the PEP binding charac- teristics of the protein. Addition of OAA in the presence of Mn 2+ to the labeled E. coli PEP carboxykinase, resulted in a 1.6-fold enhance- ment of the P-pyridoxyl fluorescence and a blue shift of 4 nm. The dissociation constant for the free ligand obtained from the fitting of the saturation curve to Eqn (2), is included in Table 1. The affinity of this substrate was also highly dependent on the nature of the cation, a negligible enhancement of fluorescence was obtained when OAA was added in the presence of Mg 2+ or in the absence of divalent cations. The OAA decarboxylation to pyruvate was lower than 12% as described in Experimental procedures. Fur- thermore, no effects on the P-pyridoxyl fluorescence were found upon pyruvate addition to P-pyridoxyl-enzyme, in the absence or presence of Mn 2+ or Mg 2+ . Binding experiments were also carried out with oxalate, an analogue of enolpyruvate, the proposed reaction inter- mediate for PEP carboxykinases [20,23,24]. The incubation of the labeled enzyme with oxalate in the presence of Mn 2+ increased the fluorescence intensity by 50%, and the emission maximum was shifted to 386 nm. The fluorescence intensity changes produced a monophasic hyperbolic saturation curve. Considering that OAA and oxalate interaction with the protein should be similar to PEP binding [25], the K d values were calculated assuming the binding of the free species, Table 1. These data show a lower affinity for the oxalate in the presence of Mg 2+ . Steady state and time resolved fluorescence quenching Time resolved emission experiments were carried in the presence of several combinations of substrates and metal ions. In all cases the decay of the fluorescence intensity of the P-pyridoxyl group fits quite well to a biexponential function (Fig. 1). Lifetimes and their fractional intensities were significantly altered only by the presence of PEP plus Mn 2+ or Mg 2+ , and the ternary combination ATP– oxalate–Mg, see Table 2. These substrate combinations caused a significant increase of the contribution of the slow component. This result points to changes in the dynamical properties of the local environment of the P-pyridoxyl group due to changes of the protein conformation induced by the binding of PEP or the ternary combination. Quenching studies of the labeled protein were per- formed with acrylamide, a polar uncharged water-soluble molecule, which can penetrate a protein matrix as a function of protein size and dynamics. Quenching experi- ments by acrylamide in the presence of several combina- tions of substrates and divalent ions at saturating concentrations were carried out by measuring the quench- ing of the static emission of the P-pyridoxyl group. The bimolecular quenching rate constants, k q , were calculated from the Stern–Volmer constants, K SV , and the amplitude average lifetimes measured for the respective metal-ligand combinations, Eqn (1). These data are given in Table 3, and show that the protein-bound P-pyridoxyl group is accessible to acrylamide, but this accessibility is lower than that of free pyridoxamine in solution. The quenching rate constant for the free pyridoxamine is in the diffusional limit control, whereas when the chromophore is bound to the enzyme, k q is threefold lower. The presence of Mn 2+ or the combined presence of substrates (or substrate analogues) and divalent cations led to a decrease of k q , as expected from the hidden of the P-pyridoxyl group in the protein matrix upon ligand binding (Fig. 4). However, the magnitude of these changes are dependent on the nature of the ligands, minor changes were found in the presence Fig. 6. Relative fluorescence changes of P-pyridoxyl-E. coli PEP carboxykinase as a function of added substrates. (A) Nucleotide- metal binding, the P-pyridoxyl-protein adduct (0.3 l M ) was titrated with increasing concen- trations of ATP (d), AMP (r), or GDP (h), inthepresenceof2m M Mn 2+ .Thelinesare fits to Eqn (2). (B) Free PEP binding, the titration of labeled protein (0.88 l M )was carried out in the presence of 1 m M Mn 2+ . The line shows the fit of the experimental data to Eqn (2). Table 2. Fluorescence lifetimes and fractional intensities of P-pyridoxyl-E. coli PEP carboxykinase in the presence of substrates and metal ions at saturating concentrations. Substrate or ligand s 1 (ns) f 1 s 2 (ns) f 2 v 2 – 5.15 0.34 1.21 0.66 1.1 ATP, Mn 2+ 5.27 0.37 1.17 0.63 1.06 PEP, Mg 2+ 5.52 0.47 1.10 0.53 1.12 PEP, Mn 2+ 6.10 0.51 1.14 0.49 1.06 ATP, oxalate, Mg 2+ 5.75 0.45 1.00 0.55 1.16 Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 4965 of metals, and the lower k q value was obtained in the presence of PEP plus Mn 2+ or Mg 2+ . The rate constants for the singlet quenching of P-pyri- doxyl bound to the enzyme in the absence of ligands were also measured by the shortened of the emission lifetimes, according to the Stern–Volmer equation: s  i =s i ¼ 1 þðk q Þ i s  i ½Qð3Þ where s° i and s i are for the emission lifetime of the component i in the absence and presence of quencher, respectively. (k q ) i is the bimolecular quenching rate constant for the i component. Values of 0.33 · 10 9 M )1 Æs )1 and 2.3 · 10 9 M )1 Æs )1 were found for the slow and fast compo- nents, respectively. The latter value is close to that found for the free pyridoxamine, and suggests that the faster lifetime decay of the P-pyridoxyl bound to the protein senses a highly exposed microenvironment. Experiments in the presence of PEP and Mn 2+ , where very important conformational changes were detected, gave (k q ) i values of 0.09 · 10 9 M )1 Æs )1 and 2.2 · 10 9 M )1 Æs )1 for the slow and the fast components, respectively. The reduced value of the quenching rate for the slow component is in agreement with the movement of the pyridoxyl chromophore towards the interior of the protein due to the presence of substrates. DISCUSSION Few K d values for enzyme–substrate complexes have been reported for ATP-dependent PEP carboxykinases. The data informed in this work for the P-pyridoxyl group bound to Lys288 of E. coli PEP carboxykinase are in good agreement with the reported values for the native enzyme (Table 1). This shows that the derivatized enzyme, even when inactive [7], retains similar affinity for the substrates. This suggests that the enzyme inactivation should be due to minor alterations in the active site region that affect catalysis but not substrate binding. On the other hand, the statisti- cal comparison between the structures of the labeled and unlabeled enzymes shows that the P-pyridoxyl group introduces almost negligible alterations in the protein structure (r.m.s. 0.95 A ˚ ). The notable fluorescence changes upon ligand binding here described show that the P-pyridoxyl group is a useful probe to monitor ligand binding and ligand-induced conformational changes in E. coli PEP carboxykinase. The molecular model of the E. coli PEP carboxykinase P-pyridoxyl adduct places the P-pyridoxyl group close to the active site region, in a position where it should not hinder substrate binding (Fig. 2). The experiments on Mn 2+ binding showed two sites for this cation, while only one low affinity site was observed for Mg 2+ . PEP-carboxykinases require divalent metal ions for catalysis. Both for GTP-dependent and ATP-dependent PEP carboxykinases, it has been described that Mg 2+ or Mn 2+ can form the active bidentate metal–nucleotide complex, while Mn 2+ is the species that binds to and activates the enzyme [3,19]. Early kinetic studies showed that the presence of millimolar concentrations of Mg 2+ and micromolar concentrations of Mn 2+ are required for optimal activity, supporting the existence of two metal ion binding sites, one for the cation–nucleotide complex, and the other for the free divalent cation [12,21]. More recent studies on the crystal structure of the ATP-Mg 2+ -Mn 2+ – pyruvate complex of E. coli PEP-carboxykinase have shown a different and high selectivity of the binding site for these divalent cations [3]. Thus, Mg 2+ or Mn 2+ can form the metal–ATP complex, while Mn 2+ has been proposed that acts as a bridge between enolpyruvate, the putative reaction intermediate, and ATP, as well as an activator of both substrates. Consequently, the lower dissociation constant for the E. coli PEP–carboxykinase– Mn 2+ complex must reflect the binding affinity of Mn 2+ to a specific site of the enzyme. A range of 23–50 l M has been reported for the dissociation constant of Mn 2+ –protein complex of ATP- and GTP-dependent PEP carboxykinases [26,27]. The high value of K d for Mg 2+ , which is similar to the second K d for Mn 2+ , could correspond to a low affinity site for the metal ions. Alternatively, the high value of K d for Mg 2+ could reflect weak binding of Mg 2+ to the specific Mn 2+ site. The influence of the divalent cation on substrate binding is markedly dependent on the substrate. Similar affinities for the corresponding metal complexes of ATP and ADP were detected in the presence of Mn 2+ or Mg 2+ , as expected from the lack of metal ion specificity for kinetic competence of metal–nucleotide complexes. The binding of OAA could be characterized only in the presence of Mn 2+ . This could be expected from the crystal structure of the E. coli PEP carboxykinase–ATP–pyruvate–Mg 2+ –Mn 2+ com- plex, which suggests that free OAA binds in the second coordination sphere of Mn 2+ [3]. Binding of CO 2 is not affected by the presence of cations, indicating that the interactions of Mn 2+ and CO 2 are independent of each other. This agrees with observations reported in GTP- dependent chicken liver PEP carboxykinase [19]. The binding affinity of free PEP to the enzyme–Mn 2+ complex was 3.2 kcalÆmol )1 higher than in the complex with Mg 2+ , and 2.8 kcalÆmol )1 more favorable than the binding affinity for ATP– or ADP–metal complexes. The dissoci- ation constant obtained for the enzyme–Mn 2+ –PEP com- plex was much lower than the K m for PEP measured by steady-state kinetics [21], and it was two orders of magni- tude lower than in the presence of Mg 2+ .Thisdramatic drop in the affinity for PEP as a result of the change of the metal ion suggests a specific role for Mn 2+ in the binding of this substrate. These facts show that even when PEP binding Table 3. Rate constants for the quenching by acrylamide of singlet excited state of P-pyridoxyl bound to the protein in the presence of different ligands at saturating concentrations. The error is estimated as ± 5% of stated values. Ligand k q (10 9 M )1 Æs )1 ) – 1.20 ATP, Mn 2+ 0.67 ATP, Mg 2+ 0.63 ATP, oxalate, Mg 2+ 0.35 ADP, Mn 2+ 0.66 AMP, Mn 2+ 0.94 Oxaloacetate, Mn 2+ 0.75 CO 2 , Mn 2+ 0.77 PEP, Mg 2+ 0.50 PEP, Mn 2+ 0.35 Mn 2+ 0.75 Pyridoxamine 3.30 4966 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in the presence of either metal ion originates changes in the protein conformation, high affinity binding is achieved only in the presence of Mn 2+ . This high binding affinity of PEP in the presence of Mn 2+ appears common to PEP- carboxykinases. A K d of 0.6 l M has been determined for the dissociation of PEP from the enzyme–Mn 2+ –PEP complex of chicken liver PEP-carboxykinase [19]. Recently, unfolding studies on the S. cerevisiae PEP-carboxykinase, a tetrameric ATP-dependent enzyme, also showed a high binding affinity of PEP in the presence of Mn 2+ [28]. The high affinity of PEP in the presence of Mn 2+ suggests a specific interaction between these two ligands in the enzyme active site. In the E. coli–ATP–pyruvate–Mg 2+ –Mn 2+ complex, Delbaere et al. [3] have shown that an oxygen atom from Pc of ATP is coordinated to enzyme-bound Mn 2+ . The results presented in this paper suggest that this interaction is conserved after the phosphoryl transfer step, and could be particularly important for PEP binding. Recently, Dunten et al. [29] found that PEP is bound to Mn 2+ through two water molecules in the human PEP carboxykinase–PEP–Mn 2+ complex. This is in agreement with our results that indicate that free PEP binds to the enzyme–metal complex. The fact that PEP binds, even with low affinity, to the E. coli carboxykinase in the presence of Mg 2+ suggests that this metal ion interacts with the enzyme at the Mn 2+ specific site thus allowing a favorable interaction of PEP with the protein. Rate constants for the quenching by acrylamide in the presence of ligands are significantly lower for the complexed than for the uncomplexed P-pyridoxyl-enzyme, indicating that a conformational change that hinders the P-pyridoxyl group in the protein occurs upon ligand binding. The molecular model based on the ATP–pyruvate–Mg 2+ – Mn 2+ complex [3] (Fig. 4) shows that substrate binding induces a conformational change that hinders the P-pyridoxyl group, which is in agreement with the acryla- mide quenching experiments. In conclusion, this study shows that the pyridoxyl chromophore of PLP is an ideal probe to detect environ- mental changes in E. coli PEP carboxykinase. The con- formational change caused by the binding of substrate, is sensed by the P-pyridoxyl group, and allowed the acquisi- tion of detailed and reliable information on the binding of several ligands and on the role of Mn 2+ and Mg 2+ on their binding. The comparison between acrylamide quenching studies and modeled structures of free and substrate bound P-pyridoxyl–E. coli PEP carboxykinase showed a very good agreement, suggesting that the labeled enzyme shifts from open (substrate-free) to closed (substrate-bound) structures upon ligand binding. 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