tailieunhanh - Báo cáo khoa học: Leishmania donovani phosphofructokinase Gene characterization, biochemical properties and structure-modelling studies

The characterization of the gene encodingLeishmania donovaniphosphofructokinase (PFK) and the biochemical properties of the expressedenzyme are . donovani has a singlePFKgene copyperhaploidgenome that encodes a polypeptide with a deducedmolecular mass of 53 988 and a pI of . The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. | Eur. J. Biochem. 269 3978-3989 2002 FEBS 2002 doi Leishmania donovani phosphofructokinase Gene characterization biochemical properties and structure-modelling studies Claudia Lóoez1 2 Nathalie Chevalier2 Veroniaue Hannaert2. Daniel J. Riaden3 Paul A. M. Michels2 . BB. BBB. and Jose Luis Ramirez1 4 1Instituto de Biologia Experimental Universidad Central de Venezuela Caracas Venezuela 2Research Unit for Tropical Diseases Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry Universite Catholique de Louvain Brussels Belgium 2CENARGENjEMBRAPA Brasilia Brazil 4Instituto de Estudios Avanzados-Ministerio de Ciencia y Tecnologia Caracas Venezuela The characterization of the gene encoding Leishmania donovani phosphofructokinase PFK and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of . The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyroppospha e PPj-dependent PFKs despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei Trypanoplasma borreli characterized in this study and a PFK found in Entamoeba histolytica. It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate fructose 6-phosphate showed slight positive cooperativity. PPi even at high concentrations did not have any effect. AMP acted as an atcií-vator of PFK shifting its kinetics for fructose 6-phosphate from slightlysigmoid to hyperbolic and increasing considerably the atlf ni ì t v for this substrate whereas GDP did not have any ehem MCd e lIngt ttudies and siae-dianraed muati-genesis were employed to shed light on the structural basis for the AMP effector specificity and