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

)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

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:
Abbreviations: OAA, oxaloacetic acid; PEP, phosphoenolpyruvate;

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

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

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

)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.

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

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-

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

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+

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

(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

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

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

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

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.

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

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

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+

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

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

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

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.
ACKNOWLEDGEMENTS
Supported by FONDECYT 1000756 and by NSERC of Canada.
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