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

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Ligand interactions and protein conformational changesof phosphopyridoxyl-labeledEscherichia coliphosphoenolpyruvatecarboxykinase determined by fluorescence spectroscopyMarı´a Victoria Encinas1, Fernando D. Gonza´lez-Nilo1, Hughes Goldie2and Emilio Cardemil11Departamento de Ciencias Quı´micas, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Chile;2Department ofMicrobiology and Immunology, University of Saskatchewan, Saskatoon, CanadaEscherichia coli phosphoenolpyruvate (PEP) carboxykinasecatalyzes the decarboxylation of oxaloacetate and transfer ofthe c-phosphoryl group of ATP to yield PEP, ADP, andCO2. The interaction of the enzyme with the substrates ori-ginates important domain movements in the protein. In thiswork, the interaction of several substrates and ligands withE. coli PEP carboxykinase has been studied in the phos-phopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatizedenzyme retained the substrate-binding characteristics of thenative protein, allowing the determination of several pro-tein–ligand dissociation constants, as well as the role ofMg2+and Mn2+in substrate binding. The binding affinityof PEP to the enzyme–Mn2+complex was )8.9 kcalÆmol)1,which is 3.2 kcalÆmol)1more favorable than in the complexwith Mg2+. For the substrate nucleotide–metal complexes,similar binding affinities ()6.0 to )6.2 kcalÆmol)1)werefound for either metal ion. The fluorescence decay of theP-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%)and 1.2 ns. These lifetimes were markedly altered in thederivatized enzyme–PEP–Mn complexes, and smallerchanges 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 consistentwith acrylamide quenching experiments, and suggest that thefluorescence changes reflect the domain movements of theprotein in solution.Keywords: Escherichia coli phosphoenolpyruvate carboxy-kinase; ligand binding; conformational changes; P-pyridoxylfluorescence spectroscopy.Escherichia coli phosphoenolpyruvate carboxykinase [PEPcarboxykinase; ATP:oxaloacetate carboxylase (trans-phos-phorylating) EC 4.1.1.49] catalyzes the reversible decarb-oxylation of oxaloacetic acid (OAA) with the associatedtransfer of the c-phosphoryl group of ATP to yield PEP andADP, where M2+is a divalent metal ion:OAA þ ATP !M2þPEP þ ADP þ CO2The physiological role of this enzyme in bacteria and mostother organisms is to catalyze the formation of PEP in thefirst committed step of gluconeogenesis [1]. The crystalstructure of free- and substrate-bound E. coli PEP carb-oxykinase has been solved at 1.9 A˚resolution [2,3]. Theenzyme is a monomeric, globular protein that belongs to thea/b protein class. The overall structure has two domains, a275 residue N-terminal domain, and a more compact 265residue C-terminal domain, with the active site in a deepcleft between them. The recently reported crystal structureof Trypanosoma cruzi PEP-carboxykinase [4] showsremarkable similarity. Upon substrate binding, the E. colienzyme undergoes a domain closure through a 20° rotationof the two domains towards each other, excluding bulksolvent from the active site and positioning active siteresidues for catalysis [3]. Results obtained with AlF3complexes of E. coli PEP carboxykinase indicate thatphosphoryl transfer occurs via a direct displacement mech-anism with associative qualities [5]. In spite of the detailedknowledge of the structural characteristics of E. coli carb-oxykinase, very little information is available for ATP-dependent carboxykinases with respect to thermodynamicdata on ligand binding [6].Chemical modification studies have shown that PLPspecifically labels the protein in a lysyl residue located atposition 288 and, upon reduction of the labeled enzyme withsodium borohydride, a P-pyridoxyl group is covalentlyattached at this site [7]. The crystal coordinates of the E. colienzyme indicate that this residue, located in the C-terminaldomain, is 9.7 A˚from Gly251, which is the closest aminoacid residue of the N-terminal domain, in the P-loop of theenzyme. Upon domain closure, the distance from Lys288 toGly251 reduces to 5.3 A˚, thus making Lys288 an excellentobservation point to follow the domain movement of theprotein in solution, provided this motion can be detected.Spectroscopic properties of the Schiff base formed uponreaction of PLP with amino acids or amines are highlydependent on medium properties such as pH or polarity[8,9]. Spectroscopic studies have been employed to obtaininformation about the mechanism of some PLP-dependentenzymes [10]. Reduction of the imine bond with NaBH4Correspondence to M. V. Encinas, Departamento de CienciasQuı´micas, Facultad de Quı´mica y Biologı´a, Universidadde Santiago de Chile, Casilla 40, Santiago 33, Chile.Fax: + 56 2 681 2108, Tel.: + 56 2 681 2575;E-mail: mencinas@lauca.usach.clAbbreviations: 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.xattaches the probe covalently to the protein, thus providinga suitable probe to detect structural conformational chan-ges, as its spectroscopic features are also expected to behighly dependent on the medium properties.In this work we took advantage of the specific labelingwith PLP of Lys288 in E. coli PEP carboxykinase to gaininsight into conformational alterations of the protein uponligand binding in solution. Analysis of the fluorescentcharacteristics of the reduced Schiff base allowed us toobtain binding constants for the interaction of severalsubstrates and ligands, and the role of metal ions on theirbinding.EXPERIMENTAL PROCEDURESMaterialsPLP, NaBH4, NaCNBH3, PEP, nucleotides, MnCl2,MgCl2, and pyridoxamine were from Sigma ChemicalCo., OAA from Boehringher Mannheim, oxalate fromMerck. Recombinant E. coli PEP carboxykinase wasobtained as described [11]. All other reagents were of thepurest commercially available grade.Labeling ofE. coliPEP carboxykinase with PLPThe enzyme (25–30 lM) was reacted with a fourfold molarexcess of PLP for 5 min at 0 °Cin50mMHepes (pH 7.5)containing 3 mMNaCNBH3. The reaction was stoppedwith 100 mMNaBH4, and excess reagents eliminated bydialysis at 4 °C against 50 mMHepes (pH 7.0). Under theseconditions, PLP specifically reacts with Lys288 [7]. Labelingstoichiometries, determined from e280¼ 67 700M)1Æcm)1for E. coli PEP carboxykinase [2,12] and e325¼9710M)1Æcm)1for the P-pyridoxyl group [8], were in therange 0.8–1.0 mol P-pyridoxyl/mol of protein.Fluorescent measurementsAll measurements were carried out at 22 °Cin50 mMHepesbuffer (pH 7.0). Steady state fluorescence measurementswere performed on a Spex Fluorolog spectrofluorometerwith excitation and emission band width of 1.25 nm. Theexcitation wavelength to follow the tryptophan fluorescencewas 295 nm, while 325 nm were used for the P-pyridoxylfluorescence. All spectra were recorded using the correctedmode. Fluorescence lifetimes were measured with anEdinburgh Instruments OB 900 time correlated singlephoton counting fluorimeter, using a hydrogen filled lampfor excitation. Fluorescence quenching experiments withacrylamide were carried out by monitoring the decrease inintensity at the emission maximum wavelength or thechange in the fluorescence lifetimes. Successive aliquots offreshly prepared solutions (5.6M)wereaddedtoacellcontaining the protein, and the respective parameter wasmeasured. Appropriate corrections were made for dilutioneffects (never exceeding 10%). The quenching data werefitted to the Stern–Volmer equation,F=F ¼ 1 þ KSV½Qð1Þwhere F ° and F are the fluorescence intensities in theabsence and presence of quencher, respectively. KSVisthe Stern–Volmer constant, which is related to thebimolecular quenching rate constant (kq) and thelifetime of the singlet excited state in the absence ofquencher (s°)byKSV¼ kqÆs°. Values of kqwere calcu-lated using the amplitude average lifetimes Æ sæ ¼ Sfisi,where fiis the amplitude fraction.The effect of ligands was analyzed by monitoring thechange of fluorescence intensity upon ligand addition toprotein solutions. OAA solutions were prepared just beforethe experiments. The decomposition of OAA under ourexperimental conditions was determined using the lacticdehydrogenase assay [13], and was lower than 12%.Concentration of CO2is expressed as total bicarbonate.The dissociation equilibrium constants (Kd)oftheprotein–ligand (LP) complexes were evaluated by the curvefitting to the quadratic equation deduced from the equilib-rium L + P , LP, considering that LP is proportional tothe emission intensity changes.YF¼ðFobsÀ FoÞ=ðF1À FoÞ¼½ðP þ L þ KdÞÀððP þ L þ KdÞ2À 4P ðLÞ1=2=2P ð2ÞWhere Fobsis the measured fluorescence intensity, Foisthe fluorescence intensity at the start of the titration, F¥is the fluorescence intensity at saturating concentrationof ligand, P the total protein concentration, and L isreferred to the ligand concentration.The distribution of metal ion as [M2+]free, [ML]and of [L]freewas calculated using the dissociation con-stants for the individual species present. The values usedwere MnÆoxalacetate ¼ 1.2 · 10)2M[14], MnÆoxa-late ¼ 1.78 · 10)4M, MgÆoxalate ¼ 4.17 · 10)3M[15]MnATP2–¼ 1.5 · 10)5M, MnHATP–¼ 2.2 · 10)3M,MgATP2–¼ 6.3 · 10)5M, MgHATP–¼ 4.8 · 10)3M,MnADP–¼ 8.1 · 10)5M, MnHADP ¼ 1.3 · 10)2M,MnAMP ¼ 4.3 · 10)3M, MnPEP–¼ 5.5 · 10)3M,MgPEP–¼ 1.8 · 10)3M[16]. For MnGDP–and MnHGDPthe values of the corresponding ADP complexes were used.The concentration of the different species at pH 7.0 wascalculated using the programCOMPLEXversion 6 (1986)written by A. Cornish-Bowden, Centre National de laRecherche Scientifique, Marseille, France.Computer-assisted three-dimensional modelingThe programsINSIGHTIIandDISCOVER972 (MSI) were usedon an O2 SGI workstation to obtain the three-dimensionalmodels of E. coli PEP carboxykinase. The structuresanalyzed were E. coli PEP carboxykinase (1OEN) [2] andthe E. coli PEP carboxykinase–Mg2+-Mn2+–ATP–pyru-vate complex (1AQ2) [3]. Amino acids lost in crystallo-graphic data were inserted into each structure. Allcalculations were carried out withDISCOVER_3 (MSI) andforce field CVFF and ESFF (MSI), that has all parametersneeded for the octahedral Mn2+coordination and aminoacids (1AQ2). This program was also employed for energyminimization and molecular dynamics. The metal ionMg2+was replaced by Mn2+in octahedral coordinationto three water molecules, a bidentate coordination to twooxygen atoms from Pband Pcof ATP, and the oxygen of thehydroxyl group of Thr255. The second Mn2+was inoctahedral coordination to two water molecules, an oxygenatom from Pcof ATP, Ne2from His232, an oxygen atomfrom the side chain of Asp269, and Nefrom Lys213.Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 4961Nefrom Lys288 of the open structure was covalently linkedto carbonyl group of PLP through an imino linkage. Then,the best position of the P-pyridoxyl group was selected byperforming an energy barrier calculation of the dihedralangle C-Ne-O-P [17], and the resulting structure was relaxedusing a cycle of simulated annealing. Initially, the systemwas gradually heated from 200 to 500 K, with increments of37.5 K for 5 ps each. Then, the system was equilibrated at500 K by 10 ps, and finally cooled to 200 K, decreasing 10 Keach 5 ps (30 steps). The relaxed structure obtained wasfinally minimized using steepest descents and conjugategradients algorithms. This final position for the P-pyridoxylgroup was used as a starting structure in the closed E. coliPEP carboxykinase model, and then it was minimized usinga simulated annealing by 200 ps. The cutoffs for van derWaals 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 siteresidues were calculated using a solvent radius of 1.4 A˚(water). For the amino acids, the fraction of solventaccessible surface area was calculated using the Gly-X-Glytripeptide model implemented inINSIGHTII[18]. The residueson either side of the index residue are mutated to glycine,and the solvent accessible surface area for the index residueis calculated (reference value for 100% solvent accessiblesurface). The reference value for each residue is dependenton the conformation of the neighboring residues.RESULTSCharacteristics of the P-pyridoxyl group linkedtoE. coliPEP carboxykinaseFree pyridoxamine, which can be considered as a model of apyridoxyl group bound to a Lys residue, exhibits anabsorption spectrum with a maximum at 326 nm at pH 7and 20 °C. This band can be assigned to the bipolar form ofthe pyridoxamine [8], as consequence of the deprotonation ofthe phenolic group. Upon excitation at 326 nm, pyridoxam-ine shows a well shaped emission band centered at 393 nm atpH 7. The absorption spectrum of the P-pyridoxyl adduct ofPEP carboxykinase exhibits a band with a maximum at326 nm due to pyridoxyl moiety and a band at 280 nmcorresponding to the aromatic amino acids of the protein.The fluorescence of the P-pyridoxyl moiety bound to theprotein at pH 7 is similar to that of free pyridoxamine, with amaximum at 393 nm. This spectral behavior reflects a highdegree of exposure of the P-pyridoxyl group to the solvent.The fluorescence decay of pyridoxamine and of theP-pyridoxyl-labeled protein were monitored at 393 nmupon excitation at 326 nm. The emission decay of pyridox-amine was monoexponential with a lifetime of 1.83 ns, whilethat of P-pyridoxyl bound to the protein could only be fittedby two exponential decays of 5.15 ns and 1.21 ns, withfractional intensities of 0.34 and 0.66, respectively (Fig. 1).This heterogeneous emission decay indicates that thepyridoxyl chromophore senses microheterogeneous envi-ronments during its lifetime due to its localized motion, torelaxation processes involving the solvent, and/or adjacentresidues on the protein surface.To get an approximation of the steric relationshipsbetween the Lys288-bound P-pyridoxyl group and theprotein structure, the corresponding complex was modeledusing the crystalline coordinates of the free E. coli PEPcarboxykinase [2]. The deviation of the resulting modelstructure (P-pyridoxyl labeled protein) from the coordinatesof the starting structure (PDB: 1OEN), give a r.m.s. value of0.95 A˚for Ca. Figure 2 shows that the P-pyridoxyl group islocated close to active site in a position that allows the accessof substrates to the active site. When the amino acid residueslocated £ 4A˚from ATP and the two metal ions wereconsidered, it was found that in the open, ligand-freestructure, the P-pyridoxyl group does not overlap anyresidue except the Thr251, which corresponds to only 4.7%of the total solvent accessible area considered. The Thr251 isa noncatalytic residue close to C2¢ of ATP. Thus, thislocation makes this chromophore a suitable probe to senseconformational changes that occur in this protein regionupon substrate binding.Effect of ligands on the P-pyridoxyl fluorescenceThe addition of metal ions or substrates to the labeledenzyme caused marked changes in the emission character-istics of the bound P-pyridoxyl group (Fig. 3). The additionof Mn2+quenched the fluorescence. However, the addi-tion of PEP or ATP in the presence of saturatingconcentrations of Mn2+increased the fluorescenceintensity. These fluorescence variations suggest thatFig. 1. Fluorescence decay profiles of P-pyridoxyl bound to the E. coliPEP carboxykinase in Hepes pH 7.0, kexc¼ 326 nm, kem¼ 393 nm (a)in absence of substrates; (b) in the presence of 1 mMPEP plus 2 mMMn2+(c) instrumental response function. The solid line corresponds toa biexponential function with s1¼ 5.15 ns (34%), s2¼ 1.21 ns for theenzyme–adduct in the absence of substrate, and s1¼ 6.10 ns (51%),and s2¼ 1.14 ns in the presence of PEP and Mn2+. Bottom: distri-bution of residuals.4962 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002conformational changes caused by the ligand are easilysensed by the P-pyridoxyl chromophore. Figure 4 shows themodeled structure of the P-pyridoxyl-PEP carboxykinase inthe free and substrate bound conformations. In the openconformation (Fig. 4A), the P-pyridoxyl moiety is rathersolvent exposed with a fractional exposed area of 0.39,meanwhile in the closed conformation (Fig. 4B) the exposedarea is 0.082, indicating that the fluorophore is now almostcompletely hindered into the protein matrix.The addition of Mn2+, an essential metal ion forcatalysis, to the labeled PEP carboxykinase caused thequenching of the fluorescence signal without any shift of thespectrum. The pattern of fluorescence quenching by Mn2+was biphasic (Fig. 5). The first phase occurred approxi-mately in the range from 0 to 0.3 mMMn2+, whereas thesecond phase implies a lower quenching that occurred atmillimolar concentrations of the metal ion. This biphasicbehavior indicates the presence of binding sites withdifferent affinities. Data of fluorescence intensity as functionof Mn2+concentration (Fig. 5) were well fitted to Eqn (2),expressed as a double binding function. Values of Kdof17.4 lMand 1.4 mMwere obtained for the high and lowaffinity binding sites, respectively (Table 1). When similarexperiments were carried out with Mg2+, fluorescencequenching was observed only at metal ion concentrations inthe millimolar range, and the data were well fitted to amonophasic saturation curve with Kdof 1.8 mM.Theseresults imply a low affinity site for the magnesium cation inthe protein.The incubation of the labeled enzyme with increasingconcentrations of adenine nucleotides in the presence ofMn2+led to a progressive enhancement of the emission,and to a blue shift of approximately 4 nm in the emissionmaximum. The increase of the fluorescence intensity gave amonophasic saturation curve. Data obtained for differentnucleotides are displayed in Fig. 6A. The dissociationconstants, calculated assuming that nucleotides bind asnucleotide–metal complex [3,19], are given in Table 1. Thesevalues show similar affinities for MnATP and MnADP, andmuch lower affinity for the metal monophosphorylatednucleotide derivative (Table 1). This is in agreement withthe expected requirement of the b-andc-phosphate groupsof the nucleotide for efficient binding to the protein active site[20]. Binding affinity of MgATP was similar to that in thepresence of Mn2+, and also similar to previous determina-tions of ATP and ADP binding to the native E. coli PEPcarboxykinase [6]. On the other hand, the addition of ATP orADP in the absence of metal ions gave emission changes atmuch higher nucleotide concentrations, suggesting bindingto low affinity, noncatalytic sites. The addition of GDP in thepresence of Mn2+to the labeled protein produced changes inthe fluorescence emission only at high concentrations(Fig. 6A), as expected from the known specificity of theE. coli PEP-carboxykinase for adenosine nucleotides [20,21].Thus, results obtained from nucleotide binding to theP-pyridoxyl-labeled enzyme, show that the enhancedemission in the presence of ADP or ATP can only be aconsequence of conformational changes caused by thebinding of the nucleotide to the enzyme active site region.Binding of CO2(expressed as total bicarbonate), anothersubstrate of the enzyme, also increased the P-pyridoxylfluorescence. The addition of this substrate to the proteinblue shifted the maximum by 5 nm and the fluorescenceFig. 3. Steady state fluorescence spectra of 2 lMP-pyridoxyl-E. coliPEP carboxykinase using kexc¼ 326 nm, in the presence of differentcombinations of substrates and metal ions: (a) in the absence of ligands;and in the presence of (b) 2 mMMn2+(c) 1 mMATP plus 2 mMMn2+(d) 0.05 mMPEP plus 1 mMMn2+.Fig. 2. Molecular model of the P-pyridoxyl-E. coli PEP carboxykinaseadduct. Nefrom Lys288 of the open structure of E. coli PEP carb-oxykinase (1OEN) is covalently linked to the carbonyl carbon of theP-pyridoxyl group (PL) through an imino linkage. The green lineshows 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) 4963increased 1.52-fold. Interestingly, CO2binding was notaffected by the presence of cations. The saturation curves inthe absence or presence of Mn2+were similar, Kdin thepresence of Mn2+was 13.7 mMwhich is close to theapparent Kmfor HCO3–(13 mM) [21]. Also, a similardissociation constant of 8.2 mMhas been determined for theenzyme–CO2complex of homologous Saccharomyces cere-visiae PEP carboxykinase [22].The addition of PEP to the labeled protein in the absenceof divalent cations produced no changes in the emissionproperties of the P-pyridoxyl chromophore. However, in thepresence of saturating concentrations of Mn2+, micromolarconcentrations of PEP produced notable changes on theP-pyridoxyl fluorescence (Fig. 6B). The intensity increasedalmost threefold and the emission maximum was blueshifted by 10 nm. The fitting of data to Eqn (2) using thefree PEP concentration gave Kdvalue of 0.25 lM(Table 1).Fluorescence data using the MnPEP concentration did notfit to Eqn (2). Binding of PEP in the presence of Mg2+wasalso accompanied by the enhancement of the fluorescenceintensity and a spectral shift of 7 nm. However, thedissociating constant was two orders of magnitude higherthan that obtained in the presence of Mn2+(Table 1).An independent estimation of the binding affinity of PEPfor E. coli PEP carboxykinase was obtained from thequenching of the emission of Trp residues of the unlabeledprotein. When the protein was titrated with PEP in thepresence of saturating concentrations of Mn2+, the intrinsicfluorescence was quenched by 10%. In spite of this smalleffect of PEP on the Trp emission, the fluorescence decreaseas function of free PEP concentration gave a saturating plotthatfittedtoEqn(2)withaKdof 0.22 lM(Table 1). Thisvalue 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 withFig. 4. Space-filling diagrams of the P-pyri-doxyl-E. coli PEP carboxykinase adduct in theopen (A) and closed (B) structures. The N-ter-minal domains of the two structures arecolored yellow, and the C-terminal domainsgreen. The phosphoryl and pyridoxyl moietiesof the P-pyridoxyl group are shown in red andmagenta, respectively. The fractional solventexposed area of the P-pyridoxyl group is 0.39and 0.082 for the open and closed structures,respectively. The molecular models for (A)and (B) are based on PDB structures 1OENand 1AQ2, respectively.Fig. 5. Changes in the fluorescence of P-pyridoxyl-E. coli PEP carb-oxykinase (3 lM)asfunctionofMn2+concentration. The solid linerepresents the fitting of data to Eqn (2) expressed as a double bindingfunction.Table 1. Dissociation equilibrium constants for the ligand-protein com-plexes.Ligand Metal ionaKd(lM)Mn2+– 17.4 ± 3.6 (40 ± 6)b1400 ± 480Mg2+– 1800 ± 380ATPMn Mn2+24 ± 1.0ATPMg Mg2+33.8 ± 2.0 (10 ± 1)bADPMn Mn2+24.8 ± 1.0 (18 ± 2)cAMPMn Mn2+128 ± 7GDPMn Mn2+508 ± 37PEP Mn2+0.24 ± 0.02 (0.21 ± 0.08)cPEP Mg2+ d53.8 ± 4.4CO2– 10 300 ± 350CO2Mn2+13 700 ± 600Oxalacetate Mn2+156 ± 17Oxalate Mn2+26 ± 2Oxalate Mg2+ d136 ± 16aMetal ion concentration was 2 mMin all cases.bFrom [6], cal-culated from the Trp fluorescence quenching in the unlabeledenzyme.cThis work, calculated from the Trp fluorescencequenching in the unlabeled enzyme.dMg2+,4mM.4964 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002P-pyridoxyl group does not alter the PEP binding charac-teristics of the protein.Addition of OAA in the presence of Mn2+to the labeledE. coli PEP carboxykinase, resulted in a 1.6-fold enhance-ment of the P-pyridoxyl fluorescence and a blue shift of4 nm. The dissociation constant for the free ligand obtainedfrom the fitting of the saturation curve to Eqn (2), isincluded in Table 1. The affinity of this substrate was alsohighly dependent on the nature of the cation, a negligibleenhancement of fluorescence was obtained when OAA wasadded in the presence of Mg2+or in the absence of divalentcations. The OAA decarboxylation to pyruvate was lowerthan 12% as described in Experimental procedures. Fur-thermore, no effects on the P-pyridoxyl fluorescence werefound upon pyruvate addition to P-pyridoxyl-enzyme, inthe absence or presence of Mn2+or Mg2+.Binding experiments were also carried out with oxalate,an analogue of enolpyruvate, the proposed reaction inter-mediate for PEP carboxykinases [20,23,24]. The incubationof the labeled enzyme with oxalate in the presence of Mn2+increased the fluorescence intensity by 50%, and theemission maximum was shifted to 386 nm. The fluorescenceintensity changes produced a monophasic hyperbolicsaturation curve. Considering that OAA and oxalateinteraction with the protein should be similar to PEPbinding [25], the Kdvalues were calculated assuming thebinding of the free species, Table 1. These data show a loweraffinity for the oxalate in the presence of Mg2+.Steady state and time resolved fluorescence quenchingTime resolved emission experiments were carried in thepresence of several combinations of substrates and metalions. In all cases the decay of the fluorescence intensity ofthe P-pyridoxyl group fits quite well to a biexponentialfunction (Fig. 1). Lifetimes and their fractional intensitieswere significantly altered only by the presence of PEP plusMn2+or Mg2+, and the ternary combination ATP–oxalate–Mg, see Table 2. These substrate combinationscaused a significant increase of the contribution of the slowcomponent. This result points to changes in the dynamicalproperties of the local environment of the P-pyridoxylgroup due to changes of the protein conformation inducedby the binding of PEP or the ternary combination.Quenching studies of the labeled protein were per-formed with acrylamide, a polar uncharged water-solublemolecule, which can penetrate a protein matrix as afunction of protein size and dynamics. Quenching experi-ments by acrylamide in the presence of several combina-tions of substrates and divalent ions at saturatingconcentrations were carried out by measuring the quench-ing of the static emission of the P-pyridoxyl group. Thebimolecular quenching rate constants, kq, were calculatedfrom the Stern–Volmer constants, KSV, and the amplitudeaverage lifetimes measured for the respective metal-ligandcombinations, Eqn (1). These data are given in Table 3, and show that the protein-bound P-pyridoxyl group isaccessible to acrylamide, but this accessibility is lower thanthat of free pyridoxamine in solution. The quenching rateconstant for the free pyridoxamine is in the diffusionallimit control, whereas when the chromophore is bound tothe enzyme, kqis threefold lower. The presence of Mn2+or the combined presence of substrates (or substrateanalogues) and divalent cations led to a decrease of kq, asexpected from the hidden of the P-pyridoxyl group in theprotein matrix upon ligand binding (Fig. 4). However, themagnitude of these changes are dependent on the natureof the ligands, minor changes were found in the presenceFig. 6. Relative fluorescence changes ofP-pyridoxyl-E. coli PEP carboxykinase as afunction of added substrates. (A) Nucleotide-metal binding, the P-pyridoxyl-protein adduct(0.3 lM) was titrated with increasing concen-trations of ATP (d), AMP (r), or GDP (h),inthepresenceof2mMMn2+.Thelinesarefits to Eqn (2). (B) Free PEP binding, thetitration of labeled protein (0.88 lM)wascarried out in the presence of 1 mMMn2+.The line shows the fit of the experimental datato Eqn (2).Table 2. Fluorescence lifetimes and fractional intensities of P-pyridoxyl-E. coli PEP carboxykinase in the presence of substrates and metal ions atsaturating concentrations.Substrate or ligand s1(ns) f1s2(ns) f2v2– 5.15 0.34 1.21 0.66 1.1ATP, Mn2+5.27 0.37 1.17 0.63 1.06PEP, Mg2+5.52 0.47 1.10 0.53 1.12PEP, Mn2+6.10 0.51 1.14 0.49 1.06ATP, oxalate, Mg2+5.75 0.45 1.00 0.55 1.16Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 4965of metals, and the lower kqvalue was obtained in thepresence of PEP plus Mn2+or Mg2+.The rate constants for the singlet quenching of P-pyri-doxyl bound to the enzyme in the absence of ligands werealso measured by the shortened of the emission lifetimes,according to the Stern–Volmer equation:si=si¼ 1 þðkqÞisi½Qð3Þwhere s°iand siare for the emission lifetime of thecomponent i in the absence and presence of quencher,respectively. (kq)iis the bimolecular quenching rate constantfor the i component. Values of 0.33 · 109M)1Æs)1and2.3 · 109M)1Æs)1were found for the slow and fast compo-nents, respectively. The latter value is close to that found forthe free pyridoxamine, and suggests that the faster lifetimedecay of the P-pyridoxyl bound to the protein senses ahighly exposed microenvironment. Experiments in thepresence of PEP and Mn2+, where very importantconformational changes were detected, gave (kq)ivalues of0.09 · 109M)1Æs)1and 2.2 · 109M)1Æs)1for the slow andthe fast components, respectively. The reduced value of thequenching rate for the slow component is in agreement withthe movement of the pyridoxyl chromophore towards theinterior of the protein due to the presence of substrates.DISCUSSIONFew Kdvalues for enzyme–substrate complexes have beenreported for ATP-dependent PEP carboxykinases. The datainformed in this work for the P-pyridoxyl group bound toLys288 of E. coli PEP carboxykinase are in good agreementwith 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 suggeststhat the enzyme inactivation should be due to minoralterations in the active site region that affect catalysisbut not substrate binding. On the other hand, the statisti-cal comparison between the structures of the labeledand unlabeled enzymes shows that the P-pyridoxylgroup introduces almost negligible alterations in the proteinstructure (r.m.s. 0.95 A˚). The notable fluorescencechanges upon ligand binding here described show that theP-pyridoxyl group is a useful probe to monitor ligandbinding and ligand-induced conformational changes inE. coli PEP carboxykinase. The molecular model of theE. coli PEP carboxykinase P-pyridoxyl adduct placesthe P-pyridoxyl group close to the active site region, in aposition where it should not hinder substrate binding(Fig. 2).The experiments on Mn2+binding showed two sites forthis cation, while only one low affinity site was observed forMg2+. PEP-carboxykinases require divalent metal ionsfor catalysis. Both for GTP-dependent and ATP-dependentPEP carboxykinases, it has been described that Mg2+orMn2+can form the active bidentate metal–nucleotidecomplex, while Mn2+is the species that binds to andactivates the enzyme [3,19]. Early kinetic studies showedthat the presence of millimolar concentrations of Mg2+andmicromolar concentrations of Mn2+are required foroptimal activity, supporting the existence of two metal ionbinding sites, one for the cation–nucleotide complex, andthe other for the free divalent cation [12,21]. More recentstudies on the crystal structure of the ATP-Mg2+-Mn2+–pyruvate complex of E. coli PEP-carboxykinase haveshown a different and high selectivity of the binding sitefor these divalent cations [3]. Thus, Mg2+or Mn2+canform the metal–ATP complex, while Mn2+has beenproposed that acts as a bridge between enolpyruvate, theputative reaction intermediate, and ATP, as well as anactivator of both substrates. Consequently, the lowerdissociation constant for the E. coli PEP–carboxykinase–Mn2+complex must reflect the binding affinity of Mn2+toa specific site of the enzyme. A range of 23–50 lMhas beenreported for the dissociation constant of Mn2+–proteincomplex of ATP- and GTP-dependent PEP carboxykinases[26,27]. The high value of Kdfor Mg2+, which is similar tothe second Kdfor Mn2+, could correspond to a low affinitysite for the metal ions. Alternatively, the high value of KdforMg2+could reflect weak binding of Mg2+to the specificMn2+site.The influence of the divalent cation on substrate bindingis markedly dependent on the substrate. Similar affinities forthe corresponding metal complexes of ATP and ADP weredetected in the presence of Mn2+or Mg2+, as expectedfrom the lack of metal ion specificity for kinetic competenceof metal–nucleotide complexes. The binding of OAA couldbe characterized only in the presence of Mn2+. This couldbe expected from the crystal structure of the E. coliPEP carboxykinase–ATP–pyruvate–Mg2+–Mn2+com-plex, which suggests that free OAA binds in the secondcoordination sphere of Mn2+[3]. Binding of CO2is notaffected by the presence of cations, indicating that theinteractions of Mn2+and CO2are independent of eachother. This agrees with observations reported in GTP-dependent chicken liver PEP carboxykinase [19].The binding affinity of free PEP to the enzyme–Mn2+complex was 3.2 kcalÆmol)1higher than in the complex withMg2+, and 2.8 kcalÆmol)1more favorable than the bindingaffinity for ATP– or ADP–metal complexes. The dissoci-ation constant obtained for the enzyme–Mn2+–PEP com-plex was much lower than the Kmfor PEP measured bysteady-state kinetics [21], and it was two orders of magni-tude lower than in the presence of Mg2+.Thisdramaticdrop in the affinity for PEP as a result of the change of themetal ion suggests a specific role for Mn2+in the binding ofthis substrate. These facts show that even when PEP bindingTable 3. Rate constants for the quenching by acrylamide of singletexcited state of P-pyridoxyl bound to the protein in the presence ofdifferent ligands at saturating concentrations. The error is estimatedas ± 5% of stated values.Ligand kq(109M)1Æs)1)– 1.20ATP, Mn2+0.67ATP, Mg2+0.63ATP, oxalate, Mg2+0.35ADP, Mn2+0.66AMP, Mn2+0.94Oxaloacetate, Mn2+0.75CO2, Mn2+0.77PEP, Mg2+0.50PEP, Mn2+0.35Mn2+0.75Pyridoxamine 3.304966 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002in the presence of either metal ion originates changes in theprotein conformation, high affinity binding is achieved onlyin the presence of Mn2+. This high binding affinity of PEPin the presence of Mn2+appears common to PEP-carboxykinases. A Kdof 0.6 lMhas been determined forthe dissociation of PEP from the enzyme–Mn2+–PEPcomplex of chicken liver PEP-carboxykinase [19]. Recently,unfolding studies on the S. cerevisiae PEP-carboxykinase, atetrameric ATP-dependent enzyme, also showed a highbinding affinity of PEP in the presence of Mn2+[28]. Thehigh affinity of PEP in the presence of Mn2+suggests aspecific interaction between these two ligands in the enzymeactive site. In the E. coli–ATP–pyruvate–Mg2+–Mn2+complex, Delbaere et al. [3] have shown that an oxygenatom from Pc of ATP is coordinated to enzyme-boundMn2+. The results presented in this paper suggest that thisinteraction 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 toMn2+through two water molecules in the human PEPcarboxykinase–PEP–Mn2+complex. This is in agreementwith our results that indicate that free PEP binds to theenzyme–metal complex. The fact that PEP binds, even withlow affinity, to the E. coli carboxykinase in the presence ofMg2+suggests that this metal ion interacts with the enzymeat the Mn2+specific site thus allowing a favorableinteraction of PEP with the protein.Rate constants for the quenching by acrylamide in thepresence of ligands are significantly lower for the complexedthan for the uncomplexed P-pyridoxyl-enzyme, indicatingthat a conformational change that hinders the P-pyridoxylgroup in the protein occurs upon ligand binding. Themolecular model based on the ATP–pyruvate–Mg2+–Mn2+complex [3] (Fig. 4) shows that substrate bindinginduces a conformational change that hinders theP-pyridoxyl group, which is in agreement with the acryla-mide quenching experiments.In conclusion, this study shows that the pyridoxylchromophore 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, issensed by the P-pyridoxyl group, and allowed the acquisi-tion of detailed and reliable information on the binding ofseveral ligands and on the role of Mn2+and Mg2+on theirbinding. The comparison between acrylamide quenchingstudies and modeled structures of free and substrate boundP-pyridoxyl–E. coli PEP carboxykinase showed a very goodagreement, suggesting that the labeled enzyme shifts fromopen (substrate-free) to closed (substrate-bound) structuresupon ligand binding.ACKNOWLEDGEMENTSSupported by FONDECYT 1000756 and by NSERC of Canada.REFERENCES1. Utter, M.F. & Kolenbrander, H.M. (1972) Formation ofoxalacetate by CO2fixation on phosphoenolpyruvate. In TheEnzymes, 3rd edn. Academic Press, New York. 1972, Vol. 6, pp. 117–168.2. Matte, A., Goldie, H., Swet, R.M. & Delbaere, L.T.J. (1996)Crystal structure of Escherichia coli phosphoenolpyruvatecarboxykinase: a new structural family with the P-loop nucleosidetriphosphate hydrolase fold. J. Mol. Biol. 256, 126–143.3. Tari, L.W., Matte, A., Goldie, A. & Delbaere, L.T.J. (1997)Mg(2+)-Mn2+ clusters in enzyme-catalyzed phosphoryl-transferreactions. Nature Struct. Biol. 4, 990–994.4. Trepani, S., Linss, J., Goldenberg, S., Fisher, H., Craievich, A.F.& Oliva, G. (2001) Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) fron Trypanosoma cruzi at2A˚resolution. J. Mol. Biol. 313, 1059–1072.5. Sudom, A.M., Prasad, L., Goldie, H. & Delbaere, L.T.J. (2001)The phosphoryl-transfer mechanism of Escherichia coli phos-phoenolpyruvate carboxykinase from the use of AlF3. J. Mol.Biol. 314, 83–92.6. Encinas, M.V., Rojas, M.C., Goldie, H. & Cardemil, E. (1993)Comparative steady-state fluorescence studies of cytosolic rat liver(GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli(ATP) phosphoenolpyruvate carboxykinases. Biochim. Biophys.Acta 1162, 195–202.7. Bazaes, S., Goldie, H., Cardemil, E. & Jabalquinto, A.M. (1995)Identification of reactive lysines in phosphoenolpyruvate carboxy-kinases from Escherichia coli and Saccharomyces cerevisiae. FEBSLett. 360, 207–210.8. Metzler, D.E. & Snell, E.E. (1955) Spectra and ionization con-stants of the vitamin B6group and related 3-hydroxypyridinederivatives. J. Am. Chem. Soc. 77, 2431–2437.9. Echeverrı´a, G.R., Catala´n, J. & Garcı´a Blanco, F. (1997) Photo-physical study of pyridoxal 5¢-phosphate and its Schiff base withn-hexylamine. Photochem. Photobiol. 66, 810–816.10. Tai, C H. & Cook, P.F. (1998) O-acetylserine sulfhydrylase. Adv.Enzymol. Rel. Areas Mol. Biol. (ed. D. L. Purich), Vol. 74 part B, pp. 185–234. John Wiley and Sons.11. 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Martel, A.E. & Smith, R.M. (1998) NIST Critically SelectedStability Constants of Metal Complexes. NIST standard referencesdatabase 46, Version 5.0.17. Gonza´lez-Nilo, F.D., Vega, R. & Cardemil, E. (2000) Mole-cular modeling of the complexes between Saccharomycescerevisiae phosphoenolpyruvate carboxykinase and the ATPanalogs pyridoxal 5¢-diphosphoadenosine and pyridoxal 5¢-tri-phosphoadenosine. Specific labeling of lysine 290. J. Prot. Chem.19, 67–73.18. Lee, B. & Richards, F.M. (1971) The interpretation of proteinstructures: estimation of static accessibility. J. Mol. Biol. 55, 379–400.19. Hebda, C.A. & Nowak, T. (1982) Phosphoenolpyruvate carboxy-kinase. Mn2+and Mn2+substrate complexes. J. Biol. Chem. 257,5515–5522.20. Matte, A., Tari, L.W., Goldie, H. & Delbaere, L.T.J. (1997)Structure and mechanism of phosphoenolpyruvate carboxykinase.J. Biol. Chem. 272, 8105–8108.Ó FEBS 2002 Ligand interactions of PEP carboxykinase (Eur. J. Biochem. 269) 496721. Krebs, A. & Bridger, W.A. (1980) The kinetic properties ofphosphoenolpyruvate carboxykinase of Escherichia coli. Can. J.Biochem. 58, 309–318.22. Herrera, L., Encinas, M.V., Jabalquinto, A.M. & Cardemil, E.(1993) Limited proteolysis of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase. J. Prot. Chem. 12, 413–418.23. Ash, D.E., Emig, F.A., Chowdhury, S.A., Satoh, Y. & Schramm, V. (1990) Mammalian and avian liver phosphoenolpyruvate car-boxykinase. Alternate substrates and inhibition by analogues ofoxaloacetate. J. Biol. Chem. 265, 7377–7384.24. Llanos, L., Briones, R., Ye´venes, A., Gonza´lez-Nilo, F.D., Frey, P.A. & Cardemil, E. (2001) Mutation Arg336 to Lys inSaccharomyces cerevisiae phosphoenolpyruvate carboxykinaseoriginates an enzyme with increased oxaloacetate decarboxylaseactivity. FEBS Lett. 493, 1–5.25. Tari, L.W., Matte, A., Pugazhenthi, U., Goldie, H. & Delbaere, T.J. (1996) Snapshot of an enzyme reaction intermediate in thestructure of the ATP-Mg2+-oxalate ternary complex of Escheri-chia coli PEP carboxykinase. Nat. Struct. Biol. 3, 355–363.26. Malebra´n, L.P. & Cardemil, E. (1987) The presence of func-tional arginine residues in phosphoenolpyruvate carboxykinasefrom Saccharomyces cerevisiae. Biochem. Biophys. Acta 915, 385–392.27. Hlavaty, J.J. & Novak, T. (2000) Characterization of the secondmetal site in avian phosphoenolpyruvate carboxykinase. Bio-chemistry 39, 1373–1388.28. Encinas, M.V., Gonza´lez-Nilo, F.D., Andreu, J.M., Alfonso, C. &Cardemil, E. (2002) Urea-induced unfolding studies of free andligand-bound tetrameric ATP-dependent Saccharomyces cerevi-siae phosphoenolpyruvate carboxykinase. Influence of quaternarystructure on protein conformational stability. Int. J. Biochem. CellBiol. 34, 1–12.29.Dunten,P.,Belunis,C.,Crowther,R.,Hollfelder,K.,Kammlott, U., Levin, W., Michel, H., Ramsey, G.B., Swain, A., Weber, D. & Wertheimer, S.J. (2002) Crystalstructure of human cytosolic phosphoenolpyruvate carboxy-kinase reveals a new GTP-binding site. J. Mol. Biol. 316,257–264.4968 M. V. Encinas et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenol pyruvate carboxykinase determined by fluorescence. carboxy-kinase; ligand binding; conformational changes; P-pyridoxyl fluorescence spectroscopy. Escherichia coli phosphoenolpyruvate carboxykinase [PEP carboxykinase;
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