title: Modulation of the enzymatic activity of papain by interdomain residues remote from the active site.
abstract: The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains. This topology suggests a possible relationship between relative domain organization and catalytic mechanism. The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues. Most modifications had no effect on enzyme activity. However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site. The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair. This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered. Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.
authors.affiliation: Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
authors.forename: D
authors.initials: D
authors.lastname: Altschuh
authors.forename: D C
authors.initials: DC
authors.lastname: Tessier
authors.forename: T
authors.initials: T
authors.lastname: Vernet
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D006639:Histidine; D010206:Papain; D003545:Cysteine