title: Multiphasic denaturation of glutathione transferase B1-1 by guanidinium chloride. Role of the dimeric structure on the flexibility of the active site.
abstract: The unfolding and refolding mechanisms of dimeric glutathione transferase GSTB1-1 from Proteus mirabilis, using guanidinium chloride as a denaturant, have been investigated. The protein transitions were monitored by enzyme activity, intrinsic fluorescence, far ultraviolet circular dichroism and gel-filtration chromatography. The non coincidence of denaturation curves at equilibrium indicates that the unfolding of GSTB1-1 is a multistep process, i. e. inactivation of the structured dimer, dissociation into partially structured monomers followed by complete unfolding. In the 50% inactivated enzyme the Km for glutathione increases threefold, while the kcat appears almost the same, indicating that the initial phase of the denaturation involves the binding site of glutathione. The rapid recovery of the folded dimer precedes the complete enzyme reactivation. This indicates that the reconstitution of the native structure of GSTB1-1 is the result of folding and association of compact monomers followed by subtle rearrangements of assembled monomers that build up the active site.
authors.affiliation: Istituto di Scienze Biochimiche, Università G. D'Annunzio, Chieti, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Sacchetta
authors.forename: A
authors.initials: A
authors.lastname: Aceto
authors.forename: T
authors.initials: T
authors.lastname: Bucciarelli
authors.forename: B
authors.initials: B
authors.lastname: Dragani
authors.forename: S
authors.initials: S
authors.lastname: Santarone
authors.forename: N
authors.initials: N
authors.lastname: Allocati
authors.forename: C
authors.initials: C
authors.lastname: Di Ilio
mesh_terms: D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D005982:Glutathione Transferase / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry*; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011513:Proteus mirabilis / Q000201:enzymology; D013050:Spectrometry, Fluorescence
chemical_list: D006146:Guanidines; D005982:Glutathione Transferase; D019791:Guanidine