Characterization of the cofactor‐independent phosphoglycerate mutase from Leishmania mexicana mexicana

Abstract

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-phosphoglycerate:The activity is inhibited by phosphate but is resistant to Cland 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 resulted 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 environment. Partial protection of His467 was observed by the addition of 25 lM CoCl 2 to the EDTA treated enzyme but not of 125 lM 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 cofactor-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 fi...

Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase

“…45 Our results showed almost 300-fold more metal than enzyme was required to reactivate the chelated PZAse in vitro under the experimental conditions that we tested. This fact was also observed in other studies with hydrolases 46,47 in contrast to other metalloenzymes that require lower concentrations of ions for their reactivation. 46 It is possible that, in vivo, the activation of a metal-depleted wild-type PZAse may not be spontaneously favorable.…”

“…This fact was also observed in other studies with hydrolases 46,47 in contrast to other metalloenzymes that require lower concentrations of ions for their reactivation. 46 It is possible that, in vivo, the activation of a metal-depleted wild-type PZAse may not be spontaneously favorable. The fact that enzymatic activity was reconstituted by titration with metal ions at unphysiologically high concentrations suggests that it is possible that assisted mechanisms to favor metal coordination by the M. tuberculosis PZAse are present in vivo.…”

Cofactor-independent Phosphoglycerate Mutase Has an Essential Role in Caenorhabditis elegans and Is Conserved in Parasitic Nematodes

“…1). They are less related to the experimentally verified iPGM enzymes from the kinetoplastid protozoans Trypanosoma brucei and Leishmania mexicana (26 -28% identical and 47-50% similar) (23,24), although the similarity extends over the full length of the proteins. (17) and the effects on C. elegans development were monitored at various time intervals following injection.…”

“…The nematode enzymes were clearly active in neutral buffer containing magnesium ions like the enzymes from E. coli (12) and T. brucei (31). However, it would be of interest in the future to evaluate the effect of other ions on activity because cobalt is preferred by the iPGM from T. brucei (31) and L. mexicana (24), whereas manganese is optimal for iPGM from Bacillus species (22,32) and present in E. coli iPGM (12).…”

Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei

“…First, the model was optimized and extended with recent information about the kinetics of enzymes (25)(26)(27)(28). Moreover, it was modified on the basis of activities of enzymes as measured in lysates of in vitro cultured growing bloodstream form trypanosomes.…”