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.
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 mm, k(cat) = 434 +/- 54 s(-1); 2-phosphoglycerate: K(m) = 0.11 +/- 0.03 mm, 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 micro m CoCl(2) to the EDTA treated enzyme but not of 125 micro m MnCl(2), suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.
Guerra
Daniel G
DG
Research Unit for Tropical Diseases,Universite, Catholique de Louvain, Brussels, Belgium.
Vertommen
Didier
D
Fothergill-Gilmore
Linda A
LA
Opperdoes
Fred R
FR
Michels
Paul A M
PA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003067:Coenzymes / Q000502:physiology*; D004492:Edetic Acid / Q000494:pharmacology; D006639:Histidine; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010736:Phosphoglycerate Mutase / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
D003067:Coenzymes; D008670:Metals; D006639:Histidine; D004492:Edetic Acid; D010736:Phosphoglycerate Mutase