tailieunhanh - Báo cáo khoa học: Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

The extremely heat-stable 5¢-methylthioadenosine phos-phorylase from the hyperthermophilic archaeonPyrococcus furiosuswas cloned, expressed to high levels inEscherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi–Bi mechanism and phosphate binding precedes nucleo-side binding in the phosphorolytic direction | Eur. J. Biochem. 271 4834-4844 2004 FEBS 2004 doi Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus Mechanism of the reaction and assignment of disulfide bonds Giovanna Cacciapuoti1 Maria Anaela Moretti2 Sabrina Forte1 Assunta Brio1. Laura Camardella3 Vincenzo Zappia1 and Marina Porcelli1 1 Dipartimento di Biochimica e Biofisica F. Cedrangolo Seconda Universita di Napoli Naples Italy 2Centro Regionale di Competenza in Biotecnologie Industriali BioTekNet Seconda Universita di Napoli Naples Italy 3Istituto di Biochimica delle Proteine CNR Naples Italy The extremely heat-stable 5 -methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned expressed to high levels in Escherichia coli and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5 -Methyl-thioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be M after 22 h incubation. This value decreases to M in the presence of 30 mM dithiothreitol furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays fluorescence measurements and SDS PAGE. The finding of multiple disulfide bridges in 5 -methylthioadenosine phosphorylase from Pyrococcus .

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