Báo cáo khoa học: Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana docx

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

convenient bacterial expression system and a high-yield
purification protocol. Kinetic properties of the bacterially
produced protein have been determined (3-phosphoglycer-
ate: K
m
¼ 0.27 ± 0.02 m
M
, k
cat
¼ 434 ± 54 s
)1
; 2-phos-
phoglycerate: K
m
¼ 0.11 ± 0.03 m
M
, k
cat
¼ 199 ± 24 s
)1
).
The activity is inhibited by phosphate but is resistant to Cl
–
and SO
4
2–
. Inactivation by EDTA is almost fully reversed by
incubation with CoCl
2
but not with MnCl

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

, suggesting that the latter
metal ion cannot be accommodated in the active site of
Leishmania PGAM.
Keywords: chemical modification; kinetics; Leishmania
mexicana; metal dependence; phosphoglycerate mutase.
The reversible isomerization of 2-phosphoglycerate (2PGA)
and 3-phosphoglycerate (3PGA) is an obligate step for both
glycolysis and gluconeogenesis. This step is carried out
in two different ways in nature, by two different types
of evolutionarily unrelated enzymes (although both
EC 5.4.2.1). The better documented enzyme is the cofac-
tor-dependent phosphoglycerate mutase (d-PGAM) due to
its requirement for 2,3-bisphophoglycerate. It is present in
some eubacteria, yeast and all vertebrates most frequently as
a dimer or tetramer of 23–30-kDa subunits [1]. The second
enzyme, called cofactor-independent phosphoglycerate
mutase (i-PGAM), is a monomeric protein of  60 kDa.
Upon comparative sequence and structure analysis, the
former enzyme has been classified as a member of
the phospho-histidine acid phosphatase superfamily [2]
and the latter as a member of the metal-dependent alkaline
phosphatase superfamily [3,4]. Whereas d-PGAM is the
enzyme present in all vertebrates, i-PGAM is found in all
plants and archaebacteria [5] and, together with d-PGAMs,
in lower eukaryotes and eubacteria [1,6].
We have previously shown that an i-PGAM participates
in glycolysis in the protist Trypanosoma brucei [7], a human
pathogen. The completely distinct structures and catalytic
mechanisms of trypanosomal and human PGAM offer
great promise for the design of inhibitors with high

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

the parasite. We have cloned the gene of this enzyme
(LmPGAM) from a genomic library, expressed it to
produce an active His-tagged protein in Escherichia coli
and purified the enzyme with a single metal affinity column.
The availability of this convenient expression and purifica-
tion system allowed us to undertake high-resolution crys-
tallographic studies [12]. We also present a detailed
biochemical characterization of the bacterially produced
enzyme and its dependency on metal ions. We studied the
accessibility of different residues involved in interactions of
the enzyme with the substrate and metal ions via chemical
modification combined with MS analysis. Our results on
irreversible inhibition strongly suggest that the design of a
substrate analogue as an irreversible inhibitor is feasible and
pave the way for the development of selective inhibitors that
may be used as lead compounds for trypanocidal drugs.
Experimental procedures
Growth, harvesting and fractionation of parasites
Promastigotes of L. mexicana mexicana strain NHOM/B2/
84/BEL46 were grown at 28 °C in the semidefined medium
SDM-79 [13], supplemented with 10% (v/v) heat-inacti-
vated foetal bovine serum (Gibco). After 4 days, cells in the
exponential phase of growth (8.7 · 10
7
cellsÆmL
)1
)were
harvested by centrifugation, washed twice in an iso-osmotic
buffer containing 3 m
M

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

3PGA to 2PGA was coupled to NADH oxidation by
lactate dehydrogenase (LDH) via enolase (ENO) and
pyruvate kinase (PYK), and following the concomitant
decrease of absorbance at 340 nm. The assay was per-
formed at 25 °C in a 1-mL reaction mixture containing
0.1
M
triethanolamine (TEA)/HCl pH 7.6, 1 m
M
MgCl
2
,
1m
M
ADP, 0.56 m
M
NADH, 0.1 m
M
CoCl
2
,1.5m
M
3PGA, and the auxiliary enzymes ENO, PYK and LDH
at final activities of 0.55, 8.0 and 13.8 UÆmL
)1
, respectively.
NADH oxidation, reverse reaction. The conversion of
2PGA to 3PGA was coupled to NADH oxidation by
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via
3-phosphoglycerate kinase (PGK). The assay was per-

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

M
3PGA into a 1 mL
quartz cuvette containing 50 m
M
Hepes pH 7.6, 0.55 U
of rabbit muscle ENO, 1 m
M
MgCl
2
,0.1m
M
CoCl
2
and
50 m
M
KCl. One activity unit (U) is defined as the
conversion of 1 lmol substrateÆmin
)1
under standard
conditions.
Auxiliary enzymes used were obtained from Roche
Molecular Biochemicals (rabbit muscle PYK and GAPDH
and yeast PGK) and Sigma (rabbit muscle ENO and bovine
heart LDH).
Measuring PGAM activity in a
Leishmania
lysate
The mutase activity was measured in 50 lLofthelysateand
different subcellular fractions by following PEP production

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.

Library screening, subcloning and sequencing
The T. brucei i-PGAM gene [7] was used as a template to
obtain a 693 bp PCR product corresponding to a generally
well conserved part of i-PGAMs (corresponding to residues
71–302 in Fig. 2). The amplified DNA was purified after
electrophoresis through agarose, labelled with
32
Pbynick
translation and used as a hybridization probe against blots
of 10 plates of E. coli infected with approximately 4000
plaque forming units per plate (approximately 15 times the
genome size) of a genomic library of L. m. mexicana
prepared in the phage vector kGEM11 [15]. Double
digestion of DNA purified from a positive phage k clone
with SacIandHindIII restriction enzymes yielded a 6 kb
fragment that hybridized with the probe. The 6 kb fragment
was ligated into plasmid pZErO-2 (Invitrogen) and used to
transform E. coli XL1-blue cells. Further digestion of the
plasmid with EcoRI gave a positive band of 3.7 kb which
was analysed by automatic sequencing using a Beckman
CEQ 2000 sequencer.
Ó FEBS 2004 Phosphoglycerate mutase of L. mexicana (Eur. J. Biochem. 271) 1799
Sequence analysis
A
BLASTP
query was performed with the newly determined
L. mexicana PGAM amino-acid sequence (LmPGAM) in
the EMBL-EBI site ( against
the SwissProt database. All i-PGAM sequences recognized
were retrieved and stored locally. Sequences corresponding

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

The entire LmPGAM gene was amplified by PCR with the
proofreading Vent DNA polymerase (New England Bio-
labs) while adding restriction sites for NcoIandXhoI at both
of its flanks. The PCR product was treated with Taq DNA
polymerase for the addition of overhanging A nucleotides to
enable insertion into the pGEM-T easy vector (Promega) by
annealing of cohesive ends. Transformed E. coli XL1-blue
cells were plated on Luria–Bertani agar with ampicillin as
selective antibiotic. The insert with the L. mexicana gene
was excised from the plasmid by double digestion with NcoI
and XhoI and ligated into a similarly treated plasmid
pET28a. The resulting plasmid pET28LmPGAM was used
to transform E. coli XL-1 blue and BL21 cells; kanamycin
was used as antibiotic for selection of recombinant clones.
The sequence appeared to be identical to the genomic
sequence determined earlier, except for a single nucleotide
difference resulting in a SfiA substitution of the second
amino acid as a consequence of the creation of the NcoI
restriction site and the presence of a tag at the C terminus
(translated as LEHHHHHH). The C-terminally tagged
protein thus produced is called C-LmPGAM. It should be
noted that a PCR fragment amplified from a genomic clone
could be used for insertion in the expression vector, because
the Leishmania i-PGAM gene does not contain any intron.
The absence of introns in protein-coding genes is a general
feature of trypanosomatids.
Production, purification and storage of protein
Optimal growth conditions were standardized in order to
obtain the highest amount of total soluble protein, as
assessed by the intensity of a 60 kDa band on SDS/

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

(10 000 g,20min,4°C). Virtually all mutase activity
measured in the supernatant was vanadate resistant and
therefore due to i-PGAM. The supernatant was then
passed through 1.5 mL of TALON (Clontech) resin
packed in a column connected to a peristaltic pump.
Fractions of  0.9 mL were collected and the protein
content was estimated by measuring absorption of UV
light at 280 nm. The column was washed with equilibra-
tion buffer and with a stepwise gradient of imidazole
using concentrations of 5, 10 and 25 m
M
.Fractions
corresponding to protein peaks were examined by SDS/
PAGE followed by Coomassie blue staining. A major
band of approximately 60 kDa appeared at 10 and
25 m
M
imidazole fractions, and were pooled separately
for further assays.
To determine optimal storage conditions of
C-LmPGAM, its specific activity as measured shortly after
TALON purification [protein in 0.1
M
TEA pH 8, 10%
(v/v) glycerol, 0.5
M
NaCl, 25 m
M
imidazole and 0.1 m
M

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

TEA pH 7.6,
0.5
M
NaCl, 20% (v/v) glycerol, 0.1 m
M
CoCl
2
;E,0.1
M
TEA pH 7.6, 0.5
M
NaCl, 20% (v/v) glycerol, 0.1 m
M
CoCl
2
,25m
M
imidazole; the protein concentration was
0.10 mgÆmL
)1
for all five conditions. The mixtures were
incubated at 4 °C, and the stability of the protein under
each condition was followed in time by regularly measuring
the activity.
Protein concentrations were measured by the Bradford
assay [17], using BSA as standard.
1800 D. G. Guerra et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Determination of kinetic parameters (
K
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

ranging from 0.03 to 0.64 m
M
. The resulting data were
similarly fitted by a Michaelis–Menten curve and linearized
plots [Lineweaver–Burk (r
2
¼ 0.9965), Hanes (r
2
¼ 0.9995)
and Eadie–Hofstee (r
2
¼ 0.9896)].
Reactivation by metals
In order to determine the time and pH dependency of
the inactivation by EDTA, aliquots of stored LmPGAM
were diluted 1 : 1 at a protein concentration of 0.2–
0.35 mgÆmL
)1
in an appropriate buffer (Mes or Hepes) to
reach pH 6.2, 7.0 or 8.0, as confirmed by indicator paper,
and incubated overnight at 4 °C. Specific activity was then
measured and found similar for each sample. Subsequently,
EDTA was added to a final concentration of 1 m
M
and the
incubation continued for different periods of time. The
incubation was stopped by diluting an aliquot 25 times in
Hepes pH 7.6, 1 m
M
dithiothreitol, 0.1 mgÆmL

)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

above, except that no CoCl
2
was added to the reaction
mixture. To determine if reactivation is immediate, another
set of reactions was performed, in which each assay was
started by the addition of the EDTA-treated enzyme to
several cuvettes each containing the complete reaction
mixture and the metal at different concentrations.
Effect of anions
The assay based on PEP production was the preferred
method for these measurements, because the use of a single
linking enzyme (ENO) resulted in a system that was easier
to interpret, especially since anions such as Cl
–
and SO
4
2–
are known to strongly affect the kinetics of rabbit muscle
PYK [18]. When examining the effect of KCl, this salt was
not added at 50 m
M
as described above for the standard
assay mixture but at variable concentrations. For the effect
of (NH
4
)
2
SO
4
, the auxiliary enzyme enolase [purchased as a

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

)were
allowedtoreactfor60minwith0.1m
M
DEPC (35-fold
molar excess). After different periods of time, 10-lL aliquots
were taken from the reaction mixture and diluted 200-fold
in ice-cold 0.1
M
Hepes (pH 7.6), 0.1 m
M
CoCl
2
and 25 m
M
imidazole to stop the reaction. A 0.12 mL sample of each
diluted aliquot was used to determine the remaining PGAM
activity by the ENO–PYK–LDH coupled assay. Two
reactions were performed by the addition of DEPC after a
5 min preincubation of the mixture at room temperature
with either 0.1 m
M
EDTA or 0.1 m
M
CoCl
2
. Only aceto-
nitrile without DEPC was added to the control samples.
Another set of reactions was performed with 8 l
M
of

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

Desalted enzyme was incubated with only acetonitrile or
allowed to react with DEPC in the presence or absence of
9m
M
3PGA. A second set of samples was first inactivated
with EDTA which was removed by centrifugation of the
protein solution through Sephadex G-50 packed in 1 mL
syringes as described [19]. The resulting desalted, EDTA-
treated enzyme was incubated for 45–60 min at 4 °Cwith
either no addition or with 25 l
M
of CoCl
2
,or125l
M
of
MnCl
2
and then treated with DEPC at room temperature
for 12 min. Specific activities were checked before and after
the filtration, EDTA treatment, CoCl
2
reactivation and
acetonitrile or DEPC treatment.
All samples were subsequently denatured by adding
1vol. 8
M
urea and incubating for 30 min; the urea
concentration was then decreased to 2
M

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

concentration even when they had coeluted with more
abundant peptides. Peptides were identified from the MS/
MS data using
TURBOSEQUEST
(ThermoFinnigan) database
search engine or manually with the help of
XCALIBUR
software (ThermoFinnigan). Search parameters incorpor-
ated a mass difference of 72.00 atomic mass units for
N-carbethoxyhistidine vs. nonmodified histidine. Abun-
dance of each peptide species was estimated by their relative
signal intensity and by their peak area after integration.
Results
PGAM activity in
L. mexicana
An initial attempt to measure the mutase activity in
Leishmania lysates was performed with the ENO–PYK–
LDH coupled assay. With this method a high background
of NADH oxidation was detected in all fractions, and the
PEP production assay was therefore preferred for locating
the mutase activity. Figure 1A shows the activity upon the
addition of 3PGA in the presence of 0.55 U of ENO and a
sample of each cell fraction. Under these conditions, the
main reaction monitored should be the PGAM- and ENO-
coupled PEP production. These experiments located the
PGAM activity in the cytosol of Leishmania, similar to
previous findings in T. brucei [7,21].
In order to attribute the activity to either a cofactor-
dependent or -independent mutase, 5 mL of cytosolic
fraction (high-speed supernatant fraction, S40) were dia-

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,

bacterially produced C-LmPGAM (production described
below). The cytosolic mutase activity showed resistance to
Na
2
VO
3
, as has been reported previously for i-PGAMs
[22,23], whereas, under similar conditions, the mammalian
enzyme was inhibited by > 90%. These data together
confirm the existence of an i-PGAM in Leishmania, present
only in the cytosol, and the absence of any detectable
d-PGAM activity in this organism.
Cloning and sequence of
Lm
PGAM
Two phage k clones of a L. mexicana genomic library
hybridized with our T. brucei i-PGAM probe and yielded
identical Southern blot results. After shortening the kDNA
by three restriction digestions, the sequencing of the
resulting 3.7-kb fragment of L. mexicana DNA that was
still recognized by the heterologous probe revealed an ORF
of 553 codons with homology to the T. brucei enzyme
(73.6% identity, Fig. 2). The predicted encoded protein
possessed a calculated molecular mass of 60 723.38 Da and
an isoelectric point of 5.26. A phylogenetic analysis clustered
the new amino acid sequence together with the T. brucei
i-PGAM and next to the enzymes of vegetal origin, while
Fig. 1. PGAM activity in L. mexicana. (A) PGAM activity in different
subcellular fractions of L. mexicana promastigotes. Activities are
expressed as total units in each fraction divided by total protein content

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

The LmPGAM gene was fused with a sequence coding for a
short His-tag at the protein’s C terminus using plasmid
pET28 for its expression in E. coli. Lysates of transformed
E. coli BL21 cells showed, upon induction of protein
production, a strong band by SDS/PAGE with the expected
molecular mass of 60 kDa that is not seen in control cells.
Approximately half of the protein appeared to be insoluble,
presumably in inclusion bodies, after conditions were
established for its optimal production in soluble form. The
soluble cell fraction was taken. A single passage through a
metal affinity column resulted in a highly pure (as assessed by
SDS/PAGE; data not shown) and active protein. In a typical
expression and purification round, a 50 mL culture yielded
14–20 mg of total soluble protein with a PGAM activity of
22 UÆmg protein
)1
. This was purified approximately 20-fold
for a final recovery of 0.9–1.2 mg of pure LmPGAM. In this
way, approximately 5–9% of all protein found in the soluble
fraction of bacterial lysates corresponded to the Leishmania
enzyme. The purified protein had a specific activity of
419±4UÆmg protein
)1
for the conversion of 3PGA to
2PGA, as measured by the NADH oxidation method.
Protein stability
NaCl, imidazole and glycerol were removed from the
purified enzyme by gel filtration to determine subsequently
the effect of different additives on its stability during storage
at 4 °C (Fig. 3A). In spite of the fact that desalting showed

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

motif according to the Pfam database (including archaebacterial enzymes).
Ó FEBS 2004 Phosphoglycerate mutase of L. mexicana (Eur. J. Biochem. 271) 1803
ENO, PYK and LDH was the preferred method for the
characterization of the forward (glycolytic) reaction,
because of the essentially irreversible nature of this assay
(–DG° 60 kJÆmol
)1
); consequently, no or little product
inhibition was observed and the maximal (initial) velocity
was maintained for a long time (2–5 min, with
SD ± 0.001). For both the forward and reverse reaction,
determination of K
m
and V
max
by direct fitting of the data
by the Michaelis–Menten equation gave virtually identical
results to those obtained from Hanes, Lineweaver–Burk or
Eadie–Hofstee plots. With regard to the forward reaction,
theenzymehasaK
m
¼ 0.27 ± 0.02 m
M
for 3PGA and a
k
cat
¼ 434 ± 54 s
)1
. For the reverse reaction, the K
m

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

M
which is 10 times lower
than in the forward one.
Effect of anions
The effect of salts on LmPGAM activity was determined for
the forward reaction (Fig. 3B). Only a minor effect of the
concentration of salts (ammonium sulphate, KCl) on
the activity was observed. Solely PO
4
3–
was able to inhibit
the reaction significantly at relatively low concentrations.
When, in a single assay in the presence of 100 m
M
potassium
phosphate, five times more substrate (25 times the K
m
instead
of five) was used, the activity was restored to 80% of the
maximum velocity (instead of 65%), reinforcing the likeli-
hood that PO
4
3–
exerts competitive inhibition. The relatively
low effect of the anions is a major difference compared to
what was observed for cofactor-dependent PGAMs, where
all ions had a considerable effect. For example, the apparent
Michaelis constants for the substrates were reported to
increase about10-foldinthe presenceof 400 m
M

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

a desalting column prior to the reactivation assays both with
Co
2+
and Mn
2+
. Also a combination of both metal ions
Fig. 3. Biochemical properties of C-LmPGAM. (A) Stability: the
activity of the enzyme was assayed after storage at 4 °C for different
periods of time in the presence of different agents. The protein con-
centration was 0.10 mgÆmL
)1
in 0.1
M
TEA pH 7.6. Additions: m,
none; h,0.5
M
NaCl; s,0.5
M
NaCl and 20% (v/v) glycerol; ·,0.5
M
NaCl, 20% glycerol and 0.1 m
M
CoCl
2
;+,0.5
M
NaCl, 20% glycerol,
0.1 m
M
CoCl

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

DEPC over protein and in the presence of 0.1 m
M
EDTA
resulted in 95% irreversible inhibition of the LmPGAM
activity, with 75% being lost in the first 5 min of incubation
(Fig. 5A). In contrast, if no EDTA was added, the
Fig. 4. Metal dependency of C-LmPGAM activity. (A) Effect of 1 m
M
EDTA with time: m,pH 6.2;j,pH7.0;d,pH 8.0;r pH 8.0, 9.3 m
M
3PGA. The inset bar diagram shows the relative values of activity
before EDTA treatment (Ctrl), after 6h 15 min at pH 8, 1 m
M
EDTA
(EDTA), and after 15 min of incubation of the EDTA treated enzyme
in the presence of 100 l
M
of MnCl
2
(Mn), FeSO
4
(Fe), CoCl
2
(Co),
NiCl
2
(Ni), CuSO
4
(Cu) or ZnCl
2

(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

M
EDTA, a double exponential fits best the first
5 min and also predicts the result at 10 min; ·, DEPC plus 1.5 m
M
3PGA;+,DEPCplus1.5m
M
PGA and 0.1 m
M
CoCl
2
. The essential
residues are protected by 3PGA (whether additional Co
2+
is present or
not); the data corresponding to both incubation with substrate and
with substrate plus cobalt were fit together by an equation for a
straight line.
Ó FEBS 2004 Phosphoglycerate mutase of L. mexicana (Eur. J. Biochem. 271) 1805
inhibitory reaction was halted after 20 min of incubation
and 40% of the original activity remained even after 1 h.
In order to compare the protective effects of different
ligands, assays were performed with a smaller excess of
DEPC to slow down the inactivation. For examining the
kinetics of the inhibition, only the first 5 min of the reaction
were taken into account, since the DEPC concentration
cannot be considered constant for longer time periods.
Irreversible inhibition of the desalted enzyme followed a
simple exponential decay for the initial 5 min of incubation
with DEPC (Fig. 5B). An attempt to fit these points by a
double-exponential curve gave an equation with two

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

average protein coverage was 60% and 14 histidine residues
out of 18 present in the enzyme could unambiguously be
identified by MS/MS fragmentation of their corresponding
peptides. First, a control sample (acetonitrile) was analysed
in order to identify the His-containing peptides. In a second
experiment, a DEPC-treated sample was analysed and the
corresponding peptides with DEPC-dependent modification
were identified taking into account a mass increase of
72.00 Da per modified residue. A total of 10 His residues
were found to be modified (Table 1), although none of them
was stoichiometrically labelled as the corresponding
unmodified peptides were still present. It should be noted
that the enzyme was fully active just before DEPC treatment
and remained stable in the presence of only acetonitrile
during the time of incubation (12 min, room temperature),
thus indicating that the observed irreversible inhibition was
entirely caused by the reaction with DEPC. In addition, two
samples were treated in the presence of a substrate (3PGA)
concentration which in earlier experiments, where PGAM
activity was assayed, showed significant protection
(100 ± 16% and 82 ± 3% of original activity). The results
are summarized in Table 1, where all histidines present in
native LmPGAM are listed. It is indicated in the table which
of these residues were modified or protected in the
experiments with DEPC and substrate + DEPC. The
location of the residues with respect to the active site or
the surface was identified by sequence alignment with the
B. stearothermophilus enzyme, of which the crystal structure
is known [35,36] and by examining a recently solved,
unpublished structure of LmPGAM (B. Poonperm, M.

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

a
Protected
a
Active site HfiA
Lys357 – – Yes n.a.
His79 + or 60 Yes 80%
His136 + – Yes 0%
His360 – – Yes 0%
His429 (M1) – – Yes Insoluble
His496 (M1) n.d. n.d. Yes 0%
His467 (M2) + (–) Yes Insoluble
His9 + + No, surface n.a.
His37 – – No, surface 100%
His47 + n.d. No, surface Not conserved
His60 + or 79 No, surface 72%
His114 + n.d. No, surface Not conserved
His125 + + No Insoluble
His148 + + No, surface Not conserved
His160 + – No Insoluble
His377 + n.d. No, surface Not conserved
a
Positive signs indicate that the modified peptide was present and its
sequence confirmed by LC-MS/MS; negative signs indicate that only
the unmodified peptide was detected. Parentheses indicate that
His467 was detected as modified but in a very low amount. n.d., A
peptide of the corresponding mass was not detected or, if detected, its
sequence was not confirmed by MS/MS analysis. n.a., Nonassayed
mutations; insoluble, cases where the mutated protein was insoluble.
1806 D. G. Guerra et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Another set of experiments was performed in which

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

encoding an amino acid sequence with 92% identity with
that of the LmPGAM reported in this paper. A similar
search using the yeast d-PGAM sequence did not yield a
significant match in the trypanosomatid databases.
The PGAM activity in cultured L. mexicana promasti-
gotes is essentially localized in the cytosolic fraction and
corresponds exclusively to a cofactor-independent enzyme,
as shown by the effects of cobalt, 2,3-bisphosphoglycerate,
EDTA and vanadate. We have developed a bacterial
expression system and purification protocol for a
LmPGAM with an eight-residue long C-terminal tag
(containing six His residues) that resulted in a yield of
 1 mg of pure protein per 50 mL of culture. Conditions for
stable storage and optimal activity assays of the enzyme
were established. The experimentally determined data were
excellently fitted by Michaelis–Menten equations, allowing
accurate calculation of the kinetic constants.
Stability assays showed that without an excess of CoCl
2
in solution, the activity of LmPGAM decreased within days.
It is important to note that passage of the enzyme through a
desalting column equilibrated with only TEA buffer did not
affect its specific activity when measured immediately
afterwards in an assay buffer containing CoCl
2
(see
Experimental procedures). However, overnight incubation
at 4 °C (or few hours at room temperature) in the presence
of EDTA led to irreversible inactivation of a major
proportion of the enzyme preparation (not shown). These

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

study. Spheres M1 and M2 correspond, respectively, to Mn1 and Mn2
in the EJJ structure. By analogy with the B. stearothermophilus
structure, M1 is proposed to be coordinated by His429 and His496,
while His467 interacts with M2. His160 is located approximately 25 A
˚
from M1 and 20 A
˚
from the bound 2PGA.
Table 2. Activity of Lm PGAM and modification of His residues in-
volved in metal binding after EDTA treatment and subsequent incubation
with Co
2+
or Mn
2+
and DEPC treatment. EDTA, remaining activities
correspond to activities after incubation with EDTA, passage through
Sephadex G-50 and, when indicated, incubation with cobalt or man-
ganese chloride; DEPC, remaining activity was measured for the co-
balt reactivated sample. Brackets indicate that His467 was found
modified in a significantly lower amount. ND, Not determined.
EDTA
EDTA +
CoCl
2
EDTA +
MnCl
2
Activity (%)
EDTA – Remaining
activity

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]

2–
stability constant equals
to 107 · 10
12
) was the inactivation virtually complete.
Notably, short incubations with EDTA were not able to
inactivate C-LmPGAM even at very high concentrations
(upto30minwith50m
M
EDTA, data not shown). These
observations are evidence of a very strong interaction of
LmPGAM with the essential metal ions.
Only cobalt was able to reverse the effects of EDTA
inactivation. More sensitive assays would be necessary to
discern if any of the tested metals was able to sustain a
mutase activity lower than 10% of the original one.
LmPGAM resembles the mutase of T. brucei [37] in
its dependence on Co
2+
. Moreover, in the case of a plant
i-PGAM, from wheat germ, it was observed that both cobalt
and manganese were able to reverse the inactivation caused
by incubation with guanidinium chloride, but the enzyme
showed a higher affinity for Co
2+
[38]. It follows that it is a
peculiarity of the trypanosomatid enzymes that this metal
cannot be replaced by Mn
2+
, which is the essential ion in the

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

higher, 76.5–300 n
M
). This, together with the apparent lack
of activity of LmPGAM with other metals, suggest that the
parasites might need a mechanism to concentrate Co
2+
in
their cytosol. Furthermore, the mutase activity of both the
bacterially produced C-LmPGAM and that in lysates of
Leishmania promastigotes was significantly increased after
an overnight incubation in the presence of CoCl
2
.This
suggests that cell lysis and the concomitant dilution of the
enzyme result in a proportion of metal-deprived enzyme.
Alternatively, the enzyme in vivo acts at less than maximal
activity due to partial occupation of the site with Co
2+
,or
competition with metal ions that render the enzyme less
active.
DEPC labelling of the enzyme resulted in the alkylation
of essential residues located in the active site rendering the
enzyme inactive. The presence of a saturating concentration
of substrate significantly hampered the inhibitory reactions
and this protection was shown to be exerted in the active site
on the residues His136 and His467. Data concerning other
active site histidines, His79 and His496, were ambiguous
since the former could not be distinguished from His60 and
the modification of the latter could not be confirmed by

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

implies that the second group of essential residues becomes
accessible only when the metal ions are removed from the
active site of the enzyme. Examining the available crystal
structure of B. stearothermophilus i-PGAM, two bound
metal ions can be observed. Coordination of the first one, in
Metal#1 site, involves His429 and His496 while the other
one,inMetal#2sitelocated4.92A
˚
away, is bound by
His467. These amino acids are conserved in every i-PGAM
(see alignment, Fig. 2). In experiments with EDTA inacti-
vated enzyme, the histidines coordinating the Metal#1 were
not modified. Even if after EDTA inactivation, the Metal#1
site is presumably also empty, it remains inaccessible for
DEPC modification leading us to conclude that this site
consists of a deeply buried set of residues. The biphasic shape
of the DEPC-reaction with the EDTA-treated enzyme
should therefore be attributed to a higher susceptibility of
the His467 of the Metal#2 coordinating site. This hypothesis
is confirmed by the partial ) albeit significant ) protection
exerted on this residue when the EDTA-treated enzyme was
incubated with 25 l
M
CoCl
2
. Importantly, five times more
MnCl
2
did not have any effect on His467 modification,
indicating that Mn

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,

neighbour to this region, was found unexpectedly modified
only in the absence of substrate, suggesting that the presence
of 3PGA protected it, in spite of being located very distant
(> 15 A
˚
) to the active site. Available enzyme–substrate
complex structures show this residue as buried, with only 1%
of its surface accessible to the solvent. Both the observations
of Rigden et al. [42] on the mutant bacterial enzyme and our
DEPC labelling results suggest that a change in conforma-
tion and/or flexibility occurs in this region in connection with
the active site. The flexibility of the substrate-free enzyme
might be higher in this region, thus increasing its accessibility
and allowing the DEPC reaction, whereas it is kept
inaccessible in the substrate–enzyme complex as shown by
the observed crystals and by our experiments with saturating
concentrations of 3PGA. We believe that due to its potential
relevance for the catalytic mechanism and drug design, the
conformation and flexibility of this region for the substrate-
free enzyme should be explored further.
Cofactor-dependent and independent PGAMs have
completely different structures, follow different reaction
mechanisms and are susceptible to differential inhibition, as
already shown by the effects of salts, vanadate and EDTA.
Highly selective anti-trypanosomatid drugs may thus be
developed from inhibitors targeting this enzyme of the
parasite.NoreactionofDEPCwiththee-amino group of
Lys357 was detected even if the possibilities of a tryptic
miscleavage and oxidation of neighbouring cysteines were
considered in the search of the corresponding modified

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

assistance, and Dr Emma Saavedra (ICP) and Buabarn Poonperm
(University of Edinburgh, Scotland) for continuous advice and
invaluable bench tips. D. G. G. acknowledges a PhD scholarship from
the ÔCommission de Coope
´
ration Universitaire au De
´
veloppement,
commission permanente du Conseil Interuniversitaire de la Commu-
naute
´
Franc¸ aiseÕ and a travel grant from the COST-B9 Action of the
European Commission for a 2-month work visit to the laboratory of
L. A. F G. in Edinburgh. D. V. is supported by the Belgian Federal
Program Interuniversity Poles of Attraction (program P5/05). This
study was financed by grants from the European Commission through
its INCO-DEV programme (contract ICA4-CT-2001-10075) to P. M.
and L. A. F G., and from the Belgian ÔFonds de la Recherche
´
Scientifique Me
´
dicaleÕ (FRSM) to P. M.
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