TAILIEUCHUNG - Báo cáo Y học: Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenol pyruvate carboxykinase determined by fluorescence spectroscopy

Escherichia coliphosphoenolpyruvate (PEP) carboxykinase catalyzes thedecarboxylationof oxaloacetateand transfer of the c-phosphoryl group of ATP to yield PEP,ADP,and CO2 . The interaction of the enzyme with the substrates ori-ginates important domainmovements in the protein. In this work,the interaction of several substrates and ligands with E. coliPEP carboxykinase has been studied in the phos-phopyridoxyl (P-pyridoxyl)-enzyme adduct. | Eur. J. Biochem. 269 - 6 0 l6 22002 FEBS 2002 doi Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoeflolpyruvate carboxykinase determined by fluorescence spectroscopy Maria Victoria Encinas1 Fernando D. Gonzalez-Nilo1 Hughes Goldie2 and Emilio Cardemil1 1Departamento de Ciencias Quimicas Facultad de Quimica y Biologia Universidad de Santiago de Chile Chile department of Microbiology and Immunology University of Saskatchewan Saskatoon Canada Escherichia coli phosphoenolpyruvate PEP carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the y-phosphoryl group of ATP to yield PEP AIPP. and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phos-phopyridoxyl P-pyridoxyl -enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein allowing the determination of several protein-ligand dissociation constants as well as the rote of Mg2 and Mn2 in substrate binding. The binding affinity of PEP to the enzyme-Mn2 complex was kcal-mol-1 which is kcal-mol-1 more favorable than in the complex with Mg2 . For the substrate nucleotide-metal complexes similar binding affinities to kcal-mol-1 were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of ns 34 and ns. These lifetimes were markedly altered in the derivatized enzyme PEP Mn complexes and smeller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open substrate-free and closed substrate-bound forms whíhi are ronsitiant with acrylamide quenching experiments and suggest

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