Equilibrium and Kinetic Unfolding Properties of Dimeric Human Glutathione Transferase A1-1

Abstract

The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

“…Secondary, tertiary and quaternary structures remained unchanged as shown by far-UV CD, fluorescence and size-exclusion HPLC respectively (results not shown). Unfolding of ∆Phe-222 with urea was reversible (recoveries in excess of 95 %) and its stability parameters [∆G(H # O) and m value] were similar to those reported for the wild-type protein [34]. The specific activities with 1-chloro-2,4-dinitrobenzene were 52p2 and 54p3 µmol\min per mg for wild-type and ∆Phe-222 respectively.…”

“…Reversibility and equilibrium unfolding studies with urea as a denaturant were performed as described previously [34]. Protein concentration was 1 µM and the concentration of urea ranged from 0 to 8 M. Structural changes were monitored by tryptophan fluorescence.…”

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

“…The binding of BSP to the human GST A1-1 was monitored by the quenching of the intrinsic protein fluorescence (excitation at 280 nm or 295 nm, emission 325 nm) and by the quenching of the AEDANS label covalently attached to Cys111. Cys111 is located in a short turn between helices A4 and A5 [23,24], and alkylation of this residue with IAEDANS does not alter the specific activity of the enzyme (with respect to CDNB as the electrophilic substrate) or the conformational stability of the enzyme [25]. It does however alter the affinity of the protein for the anionic dye ANS in that the dyes dissociation constant for the enzyme increases from 15 µM to 40 µM.…”

Human Glutathione Transferase A1-1 Demonstrates Both Half-of-the-sites and All-of-the-sites Reactivity

“…Often the half-of-the-sites reactivity noted in previous reports was associated with a change in the quaternary structure of the enzyme (such as a shift in a tetramer-dimer equilibrium) or binding of an enzyme inhibitor or activator (52)(53)(54)(55)(56)(57). In contrast, GST A1-1 does not exist in a monomeric state (58,59) and was shown in this study to exhibit different modes of active-site reactivity without the addition of inhibitors or substrate analogs. The factor(s) that determine the mode of action of this enzyme therefore remain to be elucidated.…”