title: Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.
abstract: Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.
authors.affiliation: Institut de Biologie Structurale J.-P. Ebel UMR 5075 CEA CNRS UJF, Grenoble, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Girard
authors.forename: Stéphane
authors.initials: S
authors.lastname: Marchal
authors.forename: Javier
authors.initials: J
authors.lastname: Perez
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Finet
authors.forename: Richard
authors.initials: R
authors.lastname: Kahn
authors.forename: Roger
authors.initials: R
authors.lastname: Fourme
authors.forename: Guillaume
authors.initials: G
authors.lastname: Marassio
authors.forename: Anne-Claire
authors.initials: AC
authors.lastname: Dhaussy
authors.forename: Thierry
authors.initials: T
authors.lastname: Prangé
authors.forename: Marion
authors.initials: M
authors.lastname: Giffard
authors.forename: Fabienne
authors.initials: F
authors.lastname: Dulin
authors.forename: Françoise
authors.initials: F
authors.lastname: Bonneté
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
authors.forename: Jacques H
authors.initials: JH
authors.lastname: Abraini
authors.forename: Mohamed
authors.initials: M
authors.lastname: Mezouar
authors.forename: Nathalie
authors.initials: N
authors.lastname: Colloc'h
mesh_terms: D002384:Catalysis; D006874:Hydrostatic Pressure / Q000009:adverse effects*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation / Q000528:radiation effects*; D011489:Protein Denaturation / Q000528:radiation effects*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014503:Urate Oxidase / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*
chemical_list: C469709:rasburicase; D014503:Urate Oxidase