Cysteine-specific, covalent anchoring of transition organometallic complexes to the protein papain from Carica papaya.
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.
Haquette
Pierre
P
Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Moléculaires (UMR CNRS 7576), 75231 Paris Cedex 05, France.
Salmain
Michèle
M
Svedlung
Karolina
K
Martel
Annie
A
Rudolf
Bogna
B
Zakrzewski
Janusz
J
Cordier
Stéphane
S
Roisnel
Thierry
T
Fosse
Céline
C
Jaouen
Gérard
G
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
D008301:Maleimides; D008670:Metals; D010206:Papain; D003545:Cysteine