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 wi
Trang 1Ligand interactions and protein conformational changes
carboxykinase determined by fluorescence spectroscopy
Marı´a Victoria Encinas1, Fernando D Gonza´lez-Nilo1, Hughes Goldie2and Emilio Cardemil1
1
Departamento de Ciencias Quı´micas, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Chile;2Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
Escherichia coliphosphoenolpyruvate (PEP) carboxykinase
catalyzes the decarboxylation of oxaloacetate and transfer of
the c-phosphoryl group of ATP to yield PEP,ADP,and
CO2 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
Mg2+and Mn2+in substrate binding The binding affinity
of PEP to the enzyme–Mn2+complex was)8.9 kcalÆmol)1,
which is 3.2 kcalÆmol)1more favorable than in the complex
with Mg2+ 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 coliphosphoenolpyruvate 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 M2+is a divalent metal ion:
OAAþ ATP !M2þ PEPþ ADP þ CO2
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 AlF3 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
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)
Trang 2attaches 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
E X P E R I M E N T A L P R O C E D U R E S
Materials
PLP,NaBH4,NaCNBH3,PEP,nucleotides,MnCl2,
MgCl2,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 ofE coli PEP carboxykinase with PLP
The enzyme (25–30 lM) was reacted with a fourfold molar
excess of PLP for 5 min at 0C in 50 mMHepes (pH 7.5)
containing 3 mM NaCNBH3 The reaction was stopped
with 100 mM NaBH4,and excess reagents eliminated by
dialysis at 4C against 50 mMHepes (pH 7.0) Under these
conditions,PLP specifically reacts with Lys288 [7] Labeling
stoichiometries,determined from e280¼ 67 700M )1Æcm)1
for E coli PEP carboxykinase [2,12] and e325¼
9710M )1Æcm)1for 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 22C in 50 mMHepes
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 (5.6M) were added to a cell
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=F¼ 1 þ KSV½Q ð1Þ where F and F are the fluorescence intensities in the
absence and presence of quencher,respectively KSVis
the Stern–Volmer constant,which is related to the
bimolecular quenching rate constant (kq) and the lifetime of the singlet excited state in the absence of quencher (s) by KSV¼ kqÆs Values of kqwere calcu-lated using the amplitude average lifetimes Æsæ ¼ Sfisi, where fiis 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 CO2is expressed as total bicarbonate The dissociation equilibrium constants (Kd) of the 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
YF ¼ ðFobs FoÞ=ðF1 FoÞ ¼ ½ðP þ L þ KdÞ ððP þ L þ KdÞ2 4P ðLÞ1=2=2P ð2Þ Where Fobsis the measured fluorescence intensity, Fois 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 [M2+]free,[ML] and of [L]free was calculated using the dissociation con-stants for the individual species present The values used were MnÆoxalacetate¼ 1.2 · 10)2M [14],MnÆoxa-late¼ 1.78 · 10)4M,MgÆoxalate¼ 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 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 programsINSIGHTIIandDISCOVER972 (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–Mg2+-Mn2+ –ATP–pyru-vate complex (1AQ2) [3] Amino acids lost in crystallo-graphic data were inserted into each structure All calculations were carried out withDISCOVER_3 (MSI) and force field CVFF and ESFF (MSI),that has all parameters needed for the octahedral Mn2+coordination and amino acids (1AQ2) This program was also employed for energy minimization and molecular dynamics The metal ion
Mg2+was replaced by Mn2+in octahedral coordination
to three water molecules,a bidentate coordination to two oxygen atoms from Pband Pcof ATP,and the oxygen of the hydroxyl group of Thr255 The second Mn2+ was in octahedral coordination to two water molecules,an oxygen atom from Pcof ATP,Ne2from His232,an oxygen atom from the side chain of Asp269,and N from Lys213
Trang 3Nefrom 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 inINSIGHTII[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
R E S U L T S
Characteristics of the P-pyridoxyl group linked
toE 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 20C 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 £ 4 A˚ 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 Mn2+quenched the fluorescence However,the addi-tion of PEP or ATP in the presence of saturating concentrations of Mn2+ 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.
Trang 4conformational 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 Mn2+,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 Mn2+ was biphasic (Fig 5) The first phase occurred approxi-mately in the range from 0 to 0.3 mMMn2+,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 Mn2+concentration (Fig 5) were well fitted to Eqn (2), expressed as a double binding function Values of Kd of 17.4 lM and 1.4 mM were obtained for the high and low affinity binding sites,respectively (Table 1) When similar experiments were carried out with Mg2+,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 Kd of 1.8 mM 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
Mn2+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- and c-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 Mn2+,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 Mn2+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 coliPEP-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 CO2(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 Mn2+(c) 1 m M ATP plus 2 m M Mn2+
(d) 0.05 m PEP plus 1 m Mn2+.
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.
Trang 5increased 1.52-fold Interestingly,CO2 binding was not
affected by the presence of cations The saturation curves in
the absence or presence of Mn2+were similar, Kdin the
presence of Mn2+ was 13.7 mM which is close to the
apparent Km for HCO3 (13 mM) [21] Also,a similar
dissociation constant of 8.2 mMhas been determined for the
enzyme–CO2complex of homologous Saccharomyces
cere-visiaePEP 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 Mn2+,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 Kdvalue of 0.25 lM(Table 1).
Fluorescence data using the MnPEP concentration did not
fit to Eqn (2) Binding of PEP in the presence of Mg2+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 Mn2+(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 Mn2+,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 that fitted to Eqn (2) with a Kdof 0.22 lM(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 ) as function of Mn 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 iona K d (l M )
1400 ± 480
a
Metal ion concentration was 2 m M in all cases.bFrom [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.dMg2+, 4 m M
Trang 6P-pyridoxyl group does not alter the PEP binding
charac-teristics of the protein
Addition of OAA in the presence of Mn2+to the labeled
E coliPEP 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 Mg2+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 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 incubation
of the labeled enzyme with oxalate in the presence of Mn2+
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 Kd 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 Mg2+
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
Mn2+ or Mg2+,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, kq,were calculated from the Stern–Volmer constants, KSV,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, kqis threefold lower The presence of Mn2+
or the combined presence of substrates (or substrate analogues) and divalent cations led to a decrease of kq,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),
in the presence of 2 m M Mn 2+ The lines are
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.
Trang 7of metals,and the lower kq value was obtained in the
presence 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 were
also measured by the shortened of the emission lifetimes,
according to the Stern–Volmer equation:
si=si ¼ 1 þ ðkqÞisi ½Q ð3Þ
where si and si are for the emission lifetime of the
component i in the absence and presence of quencher,
respectively (kq)iis the bimolecular quenching rate constant
for the i component Values of 0.33· 109
M )1Æs)1 and 2.3· 109
M )1Æs)1were 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 Mn2+,where very important
conformational changes were detected,gave (kq)ivalues of
0.09· 109 M )1Æs)1and 2.2· 109 M )1Æs)1for 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
D I S C U S S I O N
Few Kdvalues 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 Mn2+binding showed two sites for this cation,while only one low affinity site was observed for
Mg2+ PEP-carboxykinases require divalent metal ions for catalysis Both for GTP-dependent and ATP-dependent PEP carboxykinases,it has been described that Mg2+or
Mn2+ can form the active bidentate metal–nucleotide complex,while Mn2+ is the species that binds to and activates the enzyme [3,19] Early kinetic studies showed that the presence of millimolar concentrations of Mg2+and micromolar concentrations of Mn2+ 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-Mg2+-Mn2+– pyruvate complex of E coli PEP-carboxykinase have shown a different and high selectivity of the binding site for these divalent cations [3] Thus,Mg2+or Mn2+can form the metal–ATP complex,while Mn2+ 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–
Mn2+complex must reflect the binding affinity of Mn2+to
a specific site of the enzyme A range of 23–50 lMhas been reported for the dissociation constant of Mn2+–protein complex of ATP- and GTP-dependent PEP carboxykinases [26,27] The high value of Kdfor Mg2+,which is similar to the second Kdfor Mn2+,could correspond to a low affinity site for the metal ions Alternatively,the high value of Kdfor
Mg2+could reflect weak binding of Mg2+to the specific
Mn2+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 Mn2+ or Mg2+,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 Mn2+ This could
be expected from the crystal structure of the E coli PEP carboxykinase–ATP–pyruvate–Mg2+–Mn2+ com-plex,which suggests that free OAA binds in the second coordination sphere of Mn2+ [3] Binding of CO2 is not affected by the presence of cations,indicating that the interactions of Mn2+and CO2 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–Mn2+ complex was 3.2 kcalÆmol)1higher than in the complex with
Mg2+,and 2.8 kcalÆmol)1more favorable than the binding affinity 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 by steady-state kinetics [21],and it was two orders of magni-tude lower than in the presence of Mg2+ This dramatic drop in the affinity for PEP as a result of the change of the metal ion suggests a specific role for Mn2+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.
Trang 8in the presence of either metal ion originates changes in the
protein conformation,high affinity binding is achieved only
in the presence of Mn2+ This high binding affinity of PEP
in the presence of Mn2+ appears common to
PEP-carboxykinases A Kdof 0.6 lM has been determined for
the dissociation of PEP from the enzyme–Mn2+–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 Mn2+[28] The
high affinity of PEP in the presence of Mn2+suggests a
specific interaction between these two ligands in the enzyme
active site In the E coli–ATP–pyruvate–Mg2+–Mn2+
complex,Delbaere et al [3] have shown that an oxygen
atom from Pc of ATP is coordinated to enzyme-bound
Mn2+ 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
Mn2+ through two water molecules in the human PEP
carboxykinase–PEP–Mn2+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
Mg2+suggests that this metal ion interacts with the enzyme
at the Mn2+ 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–Mg2+–
Mn2+ 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 informaacquisi-tion on the binding of
several ligands and on the role of Mn2+and Mg2+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
A C K N O W L E D G E M E N T S
Supported by FONDECYT 1000756 and by NSERC of Canada.
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