title: Cysteine-specific, covalent anchoring of transition organometallic complexes to the protein papain from Carica papaya.
abstract: Site-directed and covalent introduction of various transition metal-organic entities to the active site of the cysteine endoproteinase, papain, was achieved by treatment of this enzyme with a series of organometallic maleimide derivatives specially designed for the purpose. Kinetic studies made it clear that time-dependent irreversible inactivation of papain occurred in the presence of these organometallic maleimides as a result of Michael addition of the sulfhydryl of Cys25. The rate and mechanism of inactivation were highly dependent on the structure of the organometallic entity attached to the maleimide group. Combined ESI-MS and IR analysis indicated that all the resulting papain adducts contained one organometallic moiety per protein molecule. This confirmed that chemospecific introduction of the metal complexes was indeed achieved. Thus, three novel reagents for heavy-atom derivatization of protein crystals, which include ruthenium, rhenium and tungsten, are now available for the introduction of electron-dense scatterers for phasing of X-ray crystallographic data.
authors.affiliation: Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Moléculaires (UMR CNRS 7576), 75231 Paris Cedex 05, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Haquette
authors.forename: Michèle
authors.initials: M
authors.lastname: Salmain
authors.forename: Karolina
authors.initials: K
authors.lastname: Svedlung
authors.forename: Annie
authors.initials: A
authors.lastname: Martel
authors.forename: Bogna
authors.initials: B
authors.lastname: Rudolf
authors.forename: Janusz
authors.initials: J
authors.lastname: Zakrzewski
authors.forename: Stéphane
authors.initials: S
authors.lastname: Cordier
authors.forename: Thierry
authors.initials: T
authors.lastname: Roisnel
authors.forename: Céline
authors.initials: C
authors.lastname: Fosse
authors.forename: Gérard
authors.initials: G
authors.lastname: Jaouen
mesh_terms: D029441:Carica / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D010206:Papain / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013055:Spectrophotometry, Infrared
chemical_list: D008301:Maleimides; D008670:Metals; D010206:Papain; D003545:Cysteine