Characterization of the cofactor-independent phosphoglycerate
mutase from
Leishmania mexicana mexicana
Histidines that coordinate the two metal ions in the active site show different
susceptibilities to irreversible chemical modification
Daniel G. Guerra
1
, Didier Vertommen
2
, Linda A. Fothergill-Gilmore
3
, Fred R. Opperdoes
1
and Paul A. M. Michels
1
1
Research Unit for Tropical Diseases, and
2
Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology
and Laboratory of Biochemistry, Universite
´
Catholique de Louvain, Brussels, Belgium;
3
Structural Biochemistry Group,
Institute of Cell and Molecular Biology, University of Edinburgh, UK
Phosphoglycerate mutase (PGAM) activity in promastigotes
of the protozoan parasite Leishmania mexicana is found only
in the cytosol. It corresponds to a cofactor-independent
PGAM as it is not stimulated by 2,3-bisphosphoglycerate
and is susceptible to EDTA and resistant to vanadate.
We have cloned and sequenced the gene and developed a

2
,FeSO
4
, CuSO
4
,
NiCl
2
or ZnCl
2
. Alkylation by diethyl pyrocarbonate resul-
ted in irreversible inhibition, but saturating concentrations of
substrate provided full protection. Kinetics of the inhibitory
reaction showed the modification of a new group of essential
residues only after removal of metal ions by EDTA. The
modified residues were identified by MS analysis of peptides
generated by trypsin digestion. Two substrate-protected
histidines in the proximity of the active site were identified
(His136, His467) and, unexpectedly, also a distant one
(His160), suggesting a conformational change in its envi-
ronment. Partial protection of His467 was observed by
the addition of 25 l
M
CoCl
2
to the EDTA treated enzyme
but not of 125 l
M
MnCl
2

selectivity for the parasite’s enzyme. Therefore, this finding
should aid in the search for new drugs that are needed
against diseases caused by members of the trypanosomatid
family (Trypanosoma, Leishmania) [8–11] for which glucose
catabolism is of vital importance.
Correspondence to P. A. M. Michels, ICP-TROP 74.39, Avenue
Hippocrate 74, B-1200 Brussels, Belgium. Fax: + 32 27626853,
Tel.: + 32 27647473, E-mail:
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
DEPC, diethyl pyrocarbonate; ENO, enolase; LDH, lactate
dehydrogenase; PEP, phosphoenolpyruvate; PGA, phosphoglycerate;
d-PGAM, cofactor-dependent phosphoglycerate mutase; i-PGAM,
cofactor-independent phosphoglycerate mutase; PGK, phospho-
glycerate kinase; PYK, pyruvate kinase; TEA, triethanolamine.
Enzymes: glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12);
enolase/2-phospho-
D
-glycerate hydrolase (EC 4.2.1.11); lactate dehy-
drogenase (EC 1.1.1.27); phosphoglycerate kinase (EC 2.7.2.3); phos-
phoglycerate mutase (EC 5.4.2.1); pyruvate kinase (EC 2.7.1.40).
Note: The novel nucleotide sequence data published here have been
deposited in the EMBL-EBI/GenBank and DDBJ databases and are
available under accession number AJ544274.
(Received 20 January 2004, revised 25 February 2004,
accepted 19 March 2004)
Eur. J. Biochem. 271, 1798–1810 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04097.x
In the study reported here we measured a PGAM activity
in lysates of cultured promastigotes (representative of the
insect-infective stage) of Leishmania mexicana, identified
it as cofactor-independent and located it to the cytosol of

imidazole (pH 7.0) and 250 m
M
sucrose, and immediately lysed by mixing to a thick paste
with silicon carbide powder previously washed with ethanol
and water, and grinding. The lysate was cleared by
centrifugation at 30 g and different cell fractions were
obtained by subsequent centrifugation steps at 1500 g;
cellular extract [S3.5] and nuclear fraction [P3.5], 5000 g;
large-granular fraction [P6.5], 15 000 g, small-granular
fraction [P11] and 140 000 g, microsomal fraction [P40]
and cytosolic fraction [S40]. As described previously [14], all
procedures were performed at 4 °C.
Enzyme assays
PGAM activity was measured by following either the
increase of UV absorbance at 240 nm due to phosphoenol-
pyruvate (PEP) production (molar extinction coefficient
1310
M
)1
Æcm
)1
) or the decrease of UV absorbance at 340 nm
due to NADH oxidation (molar extinction coefficient
6250
M
)1
Æcm
)1
) using a Beckman DU7 spectrophotometer.
NADH oxidation, forward reaction. The conversion of

formed at 25 °C in a 1-mL reaction mixture containing
0.1
M
TEA/HCl pH 7.6, 5 m
M
MgCl
2
,1m
M
dithiothreitol,
1m
M
ATP, 0.56 m
M
NADH, 0.01 m
M
CoCl
2
,0.8m
M
2PGA, and GAPDH and PGK both at 6 UÆmL
)1
.The
CoCl
2
added in the reverse reaction assay was lower in order
to avoid the formation of pink precipitates of cobalt in the
presence of dithiothreitol.
PEP production, forward reaction. The reaction was
followed upon the addition of 1.5 m

as described above. Measurements were repeated after an
overnight dialysis of all fractions at 4 °C against 200 vols of
0.1
M
Hepes pH 7.6, 0.5
M
NaCl, 25 m
M
imidazole and
0.1 m
M
CoCl
2
, in order to remove potentially interfering
metabolites. The resulting solutions were tested for mutase
activity in the presence of different potentially activating or
inhibiting compounds, in order to characterize the type
of mutase present in Leishmania:50l
M
NaVO
3
,0.6m
M
2,3-bisphosphoglycerate (Sigma) and after incubation with
5m
M
EDTA. In parallel, the bacterially produced, purified
L. mexicana i-PGAM (see below) and commercially avail-
able rabbit muscle d-PGAM (Roche Molecular Biochem-
icals) were also assayed in the presence of these compounds.

to T. brucei, Bacillus stearothermophilus and Caenorhabditis
elegans were also appended for a multiple alignment using
the program
CLUSTALX
(
BLOSUM
matrix series, default
settings). Uncorrected distances between the i-PGAM
sequences belonging to archaebacteria and all other
sequences showed values higher than 0.85 and therefore
this group was not included in any further analysis despite
their proven i-PGAM activity [5,16]. A bootstrapped
unrooted neighbour-joining tree was created with the
remaining amino acid sequences, ignoring positions with
gaps in the alignment.
An automatic alignment performed by SwissPdbViewer
between the amino acid sequences of B. stearothermo-
philus and L. mexicana was corrected manually using the
information from the
CLUSTALX
multiple alignment. Then
the L. mexicana sequence was threaded into the structures
of the B. stearothermophilus enzyme cocrystallized with
2PGA and 3PGA (PDB codes: 1EQJ and 1EJJ). The
positions of important amino acids were confirmed by
examining every residue within a 7 A
˚
radius of the 3PGA
substrate bound in the active site.
Construction of a bacterial expression system

PAGE and by total mutase activity. E. coli BL21 cells
harbouring the pET28aLmPGAM recombinant plasmid
were grown at 37 °C in 50 mL Luria–Bertani medium
with 30 lgÆmL
)1
kanamycin for approximately 4 h until
the culture reached D
600
of 0.5–0.7. Production of the
C-LmPGAM was then induced by adding isopropyl thio-
b-
D
-galactoside at a final concentration of 1 m
M
,andthe
culture was transferred to a water bath at 17 °C. After
continued growth with agitation for approximately 20 h,
the cells were harvested by centrifugation and stored at
)20 °C.
Cell pellets were resuspended in 5 mL ice cold lysis–
equilibration buffer containing 0.1
M
TEA/HCl pH 8.0,
0.5
M
NaCl, 10% (v/v) glycerol and a protease inhibitor
mixture (Roche Molecular Biochemicals) and broken by
two passages through a French pressure cell at 90 MPa.
Approximately 10 mg of protamine sulphate was mixed
with the lysate that was subsequently centrifuged

CoCl
2
] was compared with that after incubation at different
conditions. To that purpose, 4 mL of the purified protein
was concentrated approximately fourfold using a Centricon
centrifugal filter unit (Millipore) and subsequently desalted
by passing through a 5 mL Sephadex G-25 column
equilibrated with 0.1
M
TEA pH 7.6. Fractions of 0.5 mL
were taken and the absorbance at 280 nm and conductivity
measured to assess their content of protein, imidazole and
NaCl, respectively. The protein peak fractions were collec-
ted; the enzyme specific activity was checked and it appeared
essentially the same as before the treatment. The desalted
protein was then diluted 1 : 1 into five different buffers
of the following final composition: A, 0.1
M
TEA pH 7.6;
B, 0.1
M
TEA pH 7.6, 0.5
M
NaCl;C,0.1
M
TEA pH 7.6,
0.5
M
NaCl,20%(v/v)glycerol;D,0.1
M

,
k
cat
)
and pH optima
Forward reaction. To determine accurately the kinetic
constants, the concentration of 3PGA was measured
enzymatically just before the assays. For K
m
calculation,
15 assays were performed spanning a range of different
concentrations of 3PGA from 0.09 to 4.53 m
M
.The
resulting data were fitted by a hyperbolic curve according
to the Michaelis–Menten equation and evaluated by the
minimal-squares method. Evaluation was also done by
preparing linear plots according to Lineweaver–Burk (linear
regression coefficient, r
2
¼ 0.9975), Hanes (r
2
¼ 0.9986)
and Eadie–Hofstee (r
2
¼ 0.9775).
Reverse reaction. For K
m
calculation, seven assays were
performed spanning different concentrations of 2PGA

)1
BSA and
measuring its activity. The reactivation of the EDTA-
treated protein was tested by adding metal ions to the final
sample taken (pH 8.0, 6h 15 min). This was done by
addition of 100 l
M
of either MnCl
2
,FeSO
4
,CoCl
2
,NiCl
2
,
CuSO
4
or ZnCl
2
.
Reactivation by cobalt or manganese was further assayed
by incubating the EDTA-inactivated enzyme with different
concentrations of CoCl
2
or MnCl
2
for 15–30 min at room
temperature and then measuring the activity for the forward
reaction via the NADH oxidation-based assay as described

suspension in 2.8
M
(NH
4
)
2
SO
4
] was partially desalted by
removing the supernatant after a brief centrifugation prior
to the assay. In all cases the assay was started by the
addition of the C-LmPGAM.
Chemical modification of histidines
Diethyl pyrocarbonate (DEPC, Sigma) was diluted 1 : 10
in acetonitrile and stored as 1 mL aliquots at 4 °C. Its
concentration was measured by the production of
N-carbethoxyimidazole after the reaction of an aliquot with
10 m
M
imidazole and the consequent change at A
240
(3200
M
)1
Æcm
)1
).
C-LmPGAM was purified and desalted as described
above; in a 1 mL reaction, 180 lg of protein (2.9 l
M

purified and desalted protein and 50 l
M
DEPC (sixfold
molar excess) in a total volume of 0.1 mL containing 50 m
M
Hepes pH 7.6 and 250 m
M
NaCl, and PGAM inactivation
was followed for 30 min. Prior to the addition of DEPC,
these mixtures were incubated either with no addition or
in the presence of 0.1 m
M
EDTA, or 1.5 m
M
3PGA, or
0.5 m
M
CoCl
2
,or2m
M
MgCl
2
,or2m
M
MnCl
2
for 5 min
at room temperature.
Identification of modified residues

by dilution with
0.2
M
NH
4
HCO
3
and the proteins were digested overnight
with 1 lg sequencing grade trypsin at 30 °C. The digestion
was stopped by adding trifluoroacetic acid to a final
concentration of 0.1% (v/v). The peptides were analysed
using fully automated capillary LC-MS/MS. Peptides were
captured and desalted on a peptide trap (1 mm · 8mm,
Michrom Bioresources) under high flow rate conditions
(57 lLÆmin
)1
) with 1% (v/v) acetonitrile in 0.05% (v/v)
Ó FEBS 2004 Phosphoglycerate mutase of L. mexicana (Eur. J. Biochem. 271) 1801
formic acid. Separation was performed on a reversed-phase
BioBasic C18 capillary column (0.180 mm · 150 mm,
Thermo Hypersil-Keystone, Runcorn, UK). A linear
10–60% acetonitrile gradient in 0.05% aqueous formic acid
over 100 min was used at a flow rate of 3 lLÆmin
)1
after
splitting.
MS data were acquired using a LCQ Deca XP Plus ion
trap mass spectrometer (ThermoFinnigan) in data-depend-
ent MS/MS mode [20]. Dynamic exclusion enabled acqui-
sition of MS/MS spectra of peptides present at low

lyzed. A 10 kDa cut-off membrane was used to remove any
potentially interfering metabolites while preserving all
enzymes originally present in the cytosol. The specific activity
was not lowered by this deprival of any 2,3-bisphosphogly-
cerate that might have been present in the parasite’s cytosol;
on the contrary it was significantly increased, from
780 ± 6 nmolÆmin
)1
Æmg protein
)1
to 1250 ± 75 nmolÆ
min
)1
Æmg protein
)1
(Fig. 1B). This activity did not increase
when 2,3-bisphosphoglycerate was added to the assay, in
contrast to that of the mammalian d-PGAM that was
enhanced 300% by the addition of its cofactor. The increase
ofthemutaseactivityoftheLeishmania fraction might be
explained by the presence of 0.1 m
M
CoCl
2
in the dialysis
buffer, in line with the fact that i-PGAMs are metallo-
enzymes (see section ÔRequirement for metal ionsÕ below).
Further support for the parasite enzyme’s nature as a
metalloprotein is provided by the observation that its activity
is highly sensitive to EDTA, similar to that of purified,

of the lysate. S0.5, cell extract (supernatant after removal of silicon
carbide); S3.5, cellular extract; P3.5, nuclear fraction; P6.5, large-
granular fraction; P11, small-granular fraction; P40, microsomal
fraction; S40, cytosolic fraction. (B) Effect of various treatments (for a
detailed description see Experimental procedures) on the PGAM
activity in, respectively, the cytosolic (S40) fraction of L. mexicana
promastigotes, purified bacterially produced C-LmPGAM and com-
mercially available rabbit muscle d-PGAM. Dotted columns show
results before treatment and grey columns, after treatment. To assay
the effect of EDTA, the mutase was preincubated with 5 m
M
of this
compound and then diluted in the reaction mixture to a final con-
centration of 0.25 m
M
EDTA and 1 m
M
MgCl
2
in order to avoid
EDTA interfering with the (Mg
2+
-dependent) ENO activity.
1802 D. G. Guerra et al. (Eur. J. Biochem. 271) Ó FEBS 2004
having a larger distance to the bacterial ones (not shown
here, but see [7,24] and the URL quoted in the latter
reference).
Bacterial production and purification of
Lm
PGAM

no effect on LmPGAM activity when measured immedi-
ately after the elution, the activity decreased rapidly when
no stabilizer was added. The highest stabilizing effect was
observed in the presence of NaCl, CoCl
2
, imidazole or
glycerol. By comparison of the curves in Fig. 3A, we
concluded that glycerol, when present together with NaCl,
exerted some destabilizing effect. Therefore, the preferred
storage conditions included only NaCl, CoCl
2
and imida-
zole and the protein retained 80–100% of its original activity
after 1 month (data not shown).
Kinetic parameters
Kinetic constants were determined using freshly purified
and stably stored, bacterially produced protein. The meas-
urement of NADH oxidation by coupling the reaction to
Fig. 2. Multiple alignment of representative i-PGAM sequences. Residue numbering is according to the LmPGAM sequence. Annotation of
secondary structure elements is according to the B. stearothermophilus i-PGAM structure (1EJJ.pdb) and is depicted berneath the alignment:
cylinders, a-helices; arrows, b-strands. Boxes indicate amino acids conserved in all enzymes analysed (these included all the i-PGAMs annotated in
SwissProt except for the archaebacterial ones; see text). Bold, amino acids within 5 A
˚
of 3-PGA according to 1EJJ.pdb; 7 indicates amino acids
within a 7 A
˚
radius, where two substitutions are observed: B.s.A461fiL.m.S494 and B.s.E334fiL.m.Q355. Underlining indicates insertion typical
of plant and trypanosomatid i-PGAMs. The amino acids involved in chelation of metal ions are indicated with a circle d: 1, corresponding to Mn1
and 2, to Mn2 in 1EJJ.pdb. ., serine presumably involved in the phosphoenzyme intermediate. Between arrows (above the alignment, at residues
Met395, Pro501), metal-chelating motif recognized in the metalloenzyme superfamily (PFAM01676); shadowed, consensus amino acids of this

¼
0.11 ± 0.03 m
M
for 2PGA and the k
cat
¼ 199 ± 24 s
)1
.
The enzyme showed a similar pH optimum for both
directions, located between pH 7.5 and 8.2 (data not
shown). The pH–activity profile is broader for the forward
reaction with > 75% of maximal activity between pH 6.75
and 8.75. A strong sensitivity of B. megaterium i-PGAM to
low pH was reported before and shown to be related to its
interaction with essential Mn
2+
ions [25]. This was inter-
preted as a physiologically important pH-sensing mechan-
ism of the enzyme associated with its role in triggering spore
formation and germination [25,26]. The pH–activity profile
of L. mexicana i-PGAM shows effectively a very steep
slope in the range between pH 6.0 and 7.4 for the reverse
reaction. The notably higher tolerance for low pH values
observed in the forward reaction might be due to the higher
concentration of CoCl
2
used in this assay. In order to avoid
the formation of a cobalt precipitate under the reducing
conditions of the reverse reaction assay, the concentration
of this metal was kept at only 10 l

KCl [27–29].
Requirement for metal ions
Figure 4A shows the change of LmPGAM activity when
the enzyme is incubated at 4 °Cwith1m
M
EDTA for
different periods of time. Treatment with this metal chelator
inactivated the enzyme by > 90% only at pH 8.0, and the
presence of the substrate 3PGA at concentrations up to
10 m
M
showed no significant influence on this loss of
activity. The fully inactivated samples were diluted 25-fold
and incubated with different divalent metal salts. Only
CoCl
2
was able to reactivate the enzyme. A similar
experiment showed the concentration dependency of this
reactivation by cobalt and the inability of manganese to
induce the recovery of LmPGAM activity even at higher
concentrations (Fig. 4B). Notably, 1 m
M
MgCl
2
was pre-
sent in each assay, and therefore this metal ion appeared on
its own also unable to restore the mutase activity after
incubation with EDTA. The results shown in Fig. 4 were
reproduced by similar experiments where the EDTA and
EDTA–metal complexes were removed by passage through

2
and 25 m
M
imidazole. (B) Effect of anions: m,
(NH
4
)
2
SO
4
; ·,KCl;s, potassium phosphate; d, potassium phosphate
plus 6.5 m
M
3PGA (instead of 1.5 m
M
as in the standard assay).
1804 D. G. Guerra et al. (Eur. J. Biochem. 271) Ó FEBS 2004
was tested but this did not lead to higher specific activities
than obtained with cobalt ions alone.
Chemical modification of histidines
DEPC within the pH range 5.5–7.5 is reasonably specific
for reaction with histidine residues [30]. Therefore, the
irreversible carboethoxylation by DEPC has been used for
the identification of essential His residues in many different
enzymes [31,32] among which is castor plant i-PGAM [33].
Ithasalsobeenusedforthecharacterizationofhistidine-
containing metal-binding sites [34]. As DEPC also hydro-
lyses spontaneously in water, some enzyme activity may be
retained when such residues are not easily accessible for the
compound. An initial assay with a 35 · molarexcessof

(Zn). The horizontal line indicates the
background activity without enzyme. (B) Effect of MnCl
2
and CoCl
2
:
h, EDTA-treated enzyme after preincubation with MnCl
2
at the
indicated concentrations for 15–30 min at room temperature; r,
reactivation by CoCl
2
either by adding it, at different concentrations,
directly to the assay mixture without preincubation (grey line) or after
preincubating the enzyme with the metal for 15–30 min at the con-
centrations indicated (black line). All assays were performed for the
forward reaction, using the NADH oxidation method. All points are
means of replicate experiments. For incubation with CoCl
2
,fourdif-
ferent experiments were performed at different enzyme concentrations.
Fig. 5. Irreversible inhibition by diethyl pyrocarbonate. (A) Rates of
enzyme inhibition at DEPC : protein molar ratio equal to 35 (Ôfast
conditionÕ). j, Control with only acetonitrile; d,DEPCalone;s,
DEPC plus 0.1 m
M
EDTA. (B) Rate of inhibition at a DEPC : protein
molar ratio equal to 6 : 1 (Ôslow conditionÕ). d,DEPCalone,asimple
exponential curve fits the 5 first min of irreversible inhibition;
s,DEPCplus0.1m

virtually identical negative components indicating that a
simple exponential equation describes these results properly.
When the enzyme activity was monitored over periods of
10 min or longer, an arrest of the inhibitory reaction was
evident. This can be attributed to the rapid decrease of
DEPC concentration, via spontaneous hydrolysis as well as
its reaction with essential and nonessential residues. In three
more experiments that were performed in the presence of
either an excess of cobalt, magnesium, or manganese ions,
similar curves were observed with no quantitatively signi-
ficant differences (data not shown). In contrast, chelation of
divalent metal ions by incubation with EDTA made the
enzyme more susceptible to the inhibition by DEPC. In this
case, the observed results were best fitted by a double-
exponential decay curve. Both equation parameters were
negative, indicating the occurrence of two (groups of)
inhibitory reactions. The presence of 3PGA at a concentra-
tion equal to approximately five times the K
m
rendered the
enzyme virtually refractory to inactivation by DEPC. This
indicates that the residues whose modification led to
inhibition when substrate was absent are most likely
localized in the active site.
Identification of modified residues
In order to identify the active-site residues which are
susceptible to chemical modification but protected in the
presence of substrate and metal ions, we complemented the
DEPC experiments with trypsin digestion of the samples,
followed by analysis of the peptides by LC-MS/MS. The

Walkinshaw and L. A. Fothergill-Gilmore, unpublished
data). Figure 6 shows the spatial distribution of all
conserved histidines, together with two important active-
site residues, Lys357 and Ser75. All surface histidines were
modified with the sole exception of His37. In the active site,
two histidines, His136 and His467, were modified by DEPC
but protected from this reaction by the presence of
substrate, while two others, His360 and His429, were not
accessible under any condition. Interestingly, His160 was
apparently protected by the binding of 3PGA in spite of
being located far away from the active site. In the inhibited
sample, two modifications were found in the peptide
comprising both His60 and His79, whereas with substrate
present only indications for modification of a single His
were obtained. However, it was not possible to distinguish
which of these His residues was protected in the latter case.
Table 1. Modification of LmPGAM residues by DEPC and protection
by the substrate 3PGA. Histidines located closer than 10 A
˚
from the
substrate are considered as part of the active site and those with
accessibilities higher than 10% as belonging to the protein surface.
Results of site-directed mutagenesis in castor plant i-PGAM [33] are
noted aside; percentages indicate the remaining activity after HisfiAla
mutations. Histidines 53, 231 and 233 are not included since their
corresponding tryptic peptides were too small to be seen and/or
retained by the C
18
column.
Residue Inhibited

LmPGAM was incubated with EDTA followed by desalt-
ing through Sephadex G-50 columns and subsequent
addition of Co
2+
and Mn
2+
salts in order to determine
the influence of the presence of metal ions on the accessi-
bility of the active-site histidines. Table 2 shows the results
for the different EDTA-treated enzyme samples. Clearly,
the histidines corresponding to the first metal ion-binding
site (namely His429 and His496) were not modified under
any condition. His467 corresponds to the second metal
ion-binding site and it was modified to the same extent in
both samples either with no addition or with MnCl
2
.
In contrast, the sample that was partially reactivated
(54 ± 2% of original activity) by incubation with CoCl
2
showed a significant protection of His467, evidenced by a
significantly lower chromatographic peak for the corres-
ponding peptide mass.
Discussion
As shown previously for T. brucei [7], L. mexicana also
contains an i-PGAM gene. Furthermore, a
BLAST
search in
the genome database of L. major strain Friedlin (http://
www.geneDB.org/) identified on chromosome 36 an ORF

observations indicate that the enzyme contains at least one
essential metal ion that is in equilibrium between its protein-
bound and solute form, and that the metal-deprived enzyme
slowly denatures. The Co
2+
ions are, most likely, involved
in the catalytic activity (see below) but their presence seems
also important for the correct conformation of the
LmPGAM active site and consequently the stabilization
of the enzyme’s overall structure.
The stabilizing effect of imidazole, being synergistic with
the effect of CoCl
2
, underlines the importance of a soluble
cobalt reservoir. Imidazole as a metal ligand favours the
desirable 2
+
valency and hampers the irreversible formation
of Co(OH)
2
precipitates, always observable as pink dust
after a few days even at concentrations as low as 100 l
M
when no imidazole was added. It has been reported
Fig. 6. Spatial distribution of the conserved histidines in LmPGAM.
The LmPGAM sequence was threaded in the B. stearothermophilus
structure (PDB code EQJ) as described in Experimental procedures.
The substrate (product) 2PGA is displayed in thin balls-and-sticks
format, while amino acids are depicted with thick sticks. Lys357 and
Ser75 were also included in the picture because of their relevance to our

10 ± 0% 54 ± 2% 7 ± 2%
DEPC – Remaining
activity
ND 17% ND
DEPC Modification
His429 (M1) – – –
His496 (M1) – – –
His467 (M2) + (+) +
Ó FEBS 2004 Phosphoglycerate mutase of L. mexicana (Eur. J. Biochem. 271) 1807
previously that weak chelators have a synergistic effect with
manganese to reactivate B. megaterium i-PGAM [25].
The effect of EDTA was highly dependent on tempera-
ture and pH. At room temperature it may cause irreversible
inactivation as noted above, while at 4 °C a reversible
inactivation was observed, the level of which depended on
time and pH. At pH 6 and 7 (where the stability constant
of the [EDTA–Co]
2–
complex equals to 0.4 · 10
12
and
9.5 · 10
12
, respectively), 1 m
M
EDTA equilibrated with
 3 l
M
LmPGAM caused an inactivation not higher than
40%; only at pH 8 ([EDTA-Co]

bacterial i-PGAM [6,35,36,39–41]. The fact that trypanoso-
matid and plant i-PGAMs share a preference for Co
2+
is
in line with a close relationship between these mutases as
inferred from a phylogenetic analysis ([7], and our own
analysis with more sequences; data not shown) and the strong
indications that trypanosomatids have acquired many plant-
like enzymes via an alga-like endosymbiont in an ancestral
organism [24]. Further comparison of crystal structures will
possibly provide an explanation of why trypanosomatid
i-PGAM appears to require Co
2+
ions for its mutase activity
whereas the B. stearothermophilus enzyme is Mn
2+
depend-
ent. Possibly, the determinant factor will reside in different
side-chain conformations of ligating residues.
Our data strongly suggest that Co
2+
is the authentic
active-site metal ion of Leishmania PGAM, although this
remains to be proven by atomic analysis of native enzyme
purified from parasites. However, it is interesting to note
that the cobalt concentration in mammalian organisms, the
hosts of L. mexicana, is very low (8.5–66.2 n
M
in human
blood, while the range for manganese is almost 10 times

MS/MS analysis. Interestingly, Lys357 was not modified in
spite of being a presumably highly nucleophilic residue,
possibly able to withdraw a proton from Ser75, which
corresponds to Ser62 in the B. stearothermophilus enzyme,
the phosphorylated residue according to the proposed
catalytic mechanism that involves a phosphoenzyme inter-
mediate [35–37]. Previous site-directed mutagenesis studies
on the castor plant i-PGAM have shown that the conserved
histidines corresponding to LmPGAM His136 and His496
were both essential for catalytic activity, while mutating
His467 rendered the enzyme insoluble [33]. No single
modification could explain the almost complete inhibition
we observed for LmPGAM as all residues identified were
substoichiometrically labelled (our analysis did not permit
quantitative analysis of the modification stoichiometry).
However, it is most likely that the modification of either
His136 or His467 or possibly His496 is sufficient to
inactivate a molecule of LmPGAM enzyme.
The inhibition curve by DEPC without EDTA (or in the
presence of an excess of Co
2+
,Mn
2+
and Mg
2+
, data not
shown) followed in all four cases a simple exponential decay.
In contrast, in the presence of EDTA it presented a biphasic
shape, indicating the existence of two groups of essential
residues, each one being modified with different kinetics. This

2+
is not only unable to sustain
LmPGAM activity but also its accommodation in the active
site is physically unfavoured.
DEPC labelling assays are valuable because they are a
measure of the accessibility of different residues of the protein
in solution close to the physiological pH. Nowadays, the only
available solved i-PGAM crystal structures correspond to
enzyme–substrate complexes and they show the substrate
1808 D. G. Guerra et al. (Eur. J. Biochem. 271) Ó FEBS 2004
completely buried in the active site. Our data on the enzyme
in solution, showing three inaccessible histidines (His360,
His429, His496) support the observation made on the crystal
structure of an active site that is rather difficult to access for a
molecule that is about twice as large as the substrate. This is
compatible with the occurrence of an intermediate smaller
than the substrate and product (i.e. glyceric acid) which needs
to be tightly held in the active site of the phosphoenzyme until
the reaction is completed [35,36].
The existence of a small, buried active site also raises the
question of how the flexibility of the protein might create an
access way for the substrate to reach the site. Working with
B. stearothermophilus i-PGAM, Rigden et al. [42] identified
a buried region that showed alternative conformations when
Ser62 (corresponding to Ser75 in LmPGAM) was mutated to
alanine. This region consists of three residues: Leu117,
Ile146, and Tyr258, which correspond to Leu130, Val159 and
Val279 in LmPGAM according to our multiple alignment
and structure superposition (B. Poonperm, M. Walkinshaw
and L. A. Fothergill-Gilmore, unpublished data). His160,

peptide. This low reactivity might be explained if this residue
is predominantly protonated as suggested by the proposed
catalytic mechanism [35,36]. In contrast, our assays clearly
show that His467 and His136 in the active site are accessible
for an adequate inhibitor that would target these residues
for an irreversible modification. Therefore, we suggest the
design of (initially) a substrate analogue that would bear an
electrophilic group targeted to react with His467 or His136;
possibly an epoxide probe would fulfil this role as it has been
shown for a very specific irreversible inhibition of human
carbonic anhydrase II in a recent report that claims that this
group possesses the desired combination of stability and
reactivity to enable the proximity-induced coupling with the
protein surface [43].
Surprisingly, the surface-located residue His37 was never
found modified, in four independent experiments. Future
experiments should find an answer why, in LmPGAM, this
residue does not react with DEPC.
Acknowledgements
The authors thank Prof. Jacques Pe
´
rie
´
(Universite
´
Paul Sabatier,
Toulouse, France), Dr Daniel Rigden (University of Liverpool, UK)
and Dr Erkang Fan (University of Washington, Seattle, USA) for
stimulating discussions and critically reading a draft of this paper, Joris
Van Roy and Dominique Cottem (ICP, Brussels, Belgium) for technical

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