title: Ensemble perspective for catalytic promiscuity: calorimetric analysis of the active site conformational landscape of a detoxification enzyme.
abstract: Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. However, catalytic promiscuity is a poorly understood property, and no thermodynamic treatment has described the conformational landscape of promiscuous versus substrate-specific enzymes. Here, two structurally similar glutathione transferase (GST, glutathione S-transferase) isoforms with high specificity or high promiscuity are compared. Differential scanning calorimetry (DSC) indicates a reversible low temperature transition for the promiscuous GSTA1-1 that is not observed with substrate-specific GSTA4-4. This transition is assigned to rearrangement of the C terminus at the active site of GSTA1-1 based on the effects of ligands and mutations. Near-UV and far-UV circular dichroism indicate that this transition is due to repacking of tertiary contacts with the remainder of the subunit, rather than "unfolding" of the C terminus per se. Analysis of the DSC data using a modified Landau theory indicates that the local conformational landscape of the active site of GSTA1-1 is smooth, with barrierless transitions between states. The partition function of the C-terminal states is a broad unimodal distribution at all temperatures within this DSC transition. In contrast, the remainder of the GSTA1-1 subunit and the GSTA4-4 protein exhibit folded and unfolded macrostates with a significant energy barrier separating them. Their partition function includes a sharp unimodal distribution of states only at temperatures that yield either folded or unfolded macrostates. At intermediate temperatures the partition function includes a bimodal distribution. The barrierless rearrangement of the GSTA1-1 active site within a local smooth energy landscape suggests a thermodynamic basis for catalytic promiscuity.
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98177-7610.
authors: Matthew T
authors: MT
authors: Honaker
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98177-7610.
authors: Mauro
authors: M
authors: Acchione
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98177-7610.
authors: John P
authors: JP
authors: Sumida
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98177-7610. Electronic address: winky@uw.edu.
authors: William M
authors: WM
authors: Atkins
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D002384:Catalysis; D020134:Catalytic Domain; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D015233:Models, Statistical; D008968:Molecular Conformation; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D013816:Thermodynamics
chemical_list: C484555:GSTA1 protein, human; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione