Open AccessCharacterization of the cofactor‐independent phosphoglycerate mutase from Leishmania mexicana mexicana
Author(s) -
Guerra Daniel G.,
Vertommen Didier,
FothergillGilmore Linda A.,
Opperdoes Fred R.,
Michels Paul A. M.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04097.x
Subject(s) - phosphoglycerate mutase , chemistry , cofactor , biochemistry , phosphoglycerate kinase , enzyme , leishmania mexicana , enzyme kinetics , trypsin , vanadate , substrate (aquarium) , ethylenediaminetetraacetic acid , microbiology and biotechnology , active site , stereochemistry , leishmania , biology , glycolysis , chelation , inorganic chemistry , ecology , parasite hosting , world wide web , computer science
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: K m = 0.27 ± 0.02 m m , k cat = 434 ± 54 s −1 ; 2‐phosphoglycerate: K m = 0.11 ± 0.03 m m , k cat = 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 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 µ m CoCl 2 to the EDTA treated enzyme but not of 125 µ m MnCl 2 , suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.