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: michels@bchm.ucl.ac.be 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 (http://www.ebi.ac.uk/blast2/) 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 [...]... Krishnasamy, G (2000) Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus EMBO J 19, 1419–1431 Jedrzejas, M.J., Chander, M., Setlow, P & Krishnasamy, G (2000) Mechanism of catalysis of the cofactor-independent phosphoglycerate mutase from Bacillus stearothermophilus Crystal structure of the complex with 2 -phosphoglycerate J Biol Chem 275, 23146–23153... 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 Ó FEBS 2004 Phosphoglycerate mutase of L mexicana (Eur J Biochem 271) 1809 completely buried in the active site Our data on the enzyme in... to concentrate Co2+ 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 CoCl2 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... 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 No reaction of DEPC with the e-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... (1996) Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species J Bacteriol 178, 2204–2210 27 Rose, Z.B & Dube, S (1978) Phosphoglycerate mutase Kinetics and effects of salts on the mutase and bisphosphoglycerate 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 phosphatase activities of the enzyme from chicken breast... occupation of the site with Co2+, or competition with metal ions that render the enzyme less active Ó FEBS 2004 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... phosphomutase from spores and cells of Bacillus megaterium J Bacteriol 137, 1024–1027 Singh, R.P & Setlow, P (1979) Regulation of phosphoglycerate phosphomutase in developing forespores and dormant and germinated spores of Bacillus megaterium by the level of free manganous ions J Bacteriol 139, 889–898 Chander, M., Setlow, B & Setlow, P (1998) The enzymatic activity of phosphoglycerate mutase from gram-positive... 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 Co2+ 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... 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... 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 nM in human blood, while the range for manganese is almost 10 times higher, 76.5–300 nM) This, together with the apparent lack of activity of LmPGAM with other metals, suggest that the parasites might need . Characterization of the cofactor-independent phosphoglycerate mutase from Leishmania mexicana mexicana Histidines that coordinate the two metal. Mechanism of catalysis of the cofactor-independent phos- phoglycerate mutase from Bacillus stearothermophilus.Crystal structure of the complex with 2 -phosphoglycerate.