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

Phosphoglyceratemutase (PGAM) activity inpromastigotes of theprotozoanparasiteLeishmaniamexicanais foundonly 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:Km¼ ± mM, kcat ¼434 ± 54 s )1 ; 2-phos-phoglycerate:Km¼ ± mM,kcat ¼199 ± 24 s )1 ). The activity is inhibited by phosphate but is resistant to Cl – andSO4 2– . . | Eur. J. Biochem. 271 1798-1810 2004 FEBS 2004 doi 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. Guerra1 Didier Vertommen2 Linda A. Fothergill-Gilmore3 Fred R. Opperdoes1 and Paul A. M. Michels1 1 Research Unit for Tropical Diseases and 2Hormone and Metabolic Research Unit Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry Universite Catholique de Louvain Brussels Belgium 3Structural 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 Km mM kcat 434 54 s-1 2-phos-phoglycerate Km mM kcat 199 24 s-1 . The activity is inhibited by phosphate but is resistant to Cl-and SO42-. Inactivation by EDTA is almost fully reversed by incubation with CoCl2 but not with MnCl2 FeSO4 CuSO4 NiCl2 or ZnCl2. 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 .

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