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Cysteine‐Specific, Covalent Anchoring of Transition Organometallic Complexes to the Protein Papain from Carica papaya
Author(s) -
Haquette Pierre,
Salmain Michèle,
Svedlung Karolina,
Martel Annie,
Rudolf Bogna,
Zakrzewski Janusz,
Cordier Stéphane,
Roisnel Thierry,
Fosse Céline,
Jaouen Gérard
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600387
Subject(s) - papain , chemistry , maleimide , covalent bond , rhenium , moiety , cysteine , stereochemistry , carborane , polymer chemistry , combinatorial chemistry , organic chemistry , enzyme
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.