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Conformational Heterogeneity and Catalytic Promiscuity in Glutathione Transferases

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Conformational Heterogeneity and Catalytic Promiscuity in Glutathione Transferases Conformational Heterogeneity and Catalytic Promiscuity in Glutathione Transferases Matthew T. Honaker A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2012 Reading Committee: William M. Atkins, Chair Carlos Catalano Kelly Lee Program Authorized to Offer Degree: Medicinal Chemistry University of Washington Abstract Conformational Heterogeneity and Catalytic Promiscuity in Glutathione Transferases Matthew T. Honaker Chair of the Supervisory Committee: Professor William M. Atkins Department of Medicinal Chemistry Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. Detoxification enzymes, such as glutathione transferases, are known to be highly promiscuous. One common suggestion is that promiscuous enzymes are more conformationally heterogeneous than their substrate-specific counterparts. Here, a series of structurally similar glutathione transferase (GST) variants ranging from high specificity to high promiscuity are used as a platform for examining conformational heterogeneity as a putative mechanism for promiscuity. Quantitative promiscuity indices range from 0.72 for promiscuous GSTA1-1 to 0.05 for specific GSTA4-4, with intermediate values for each of the GST mutants. Fluorescence lifetime distribution analysis of Trp21 at the domain interface indicates a modest correlation of core protein dynamics with promiscuity. The mobility during the excited-state lifetime of Trp21 increases sharply for GSTA1-1. 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. Extraction and analysis of low temperature transitions for GSTA4-4 and the GST mutants with intermediate promiscuity using the same method indicates a correlation between the natural logarithm of partition function distribution widths with promiscuity, suggestive of an entropic basis for promiscuity. A similar trend in barrier heights differentiated barrierless GSTA1-1 from the less promiscuous GSTs, with free energy barriers increasing as promiscuity decreased. The correlation of conformational heterogeneity and level of promiscuity across the GST variants examined suggest a conformational selection mechanism for catalytic promiscuity. Table of Contents Page List of Figures ................................................................................................................................ iii List of Tables ................................................................................................................................. iv Chapter 1: Introduction and Background ........................................................................................1 A. Introduction ............................................................................................................................ 1 B. Ensemble Perspective ............................................................................................................. 2 C. Promiscuity............................................................................................................................. 8 D. Glutathione Transferases ...................................................................................................... 15 Chapter 2: Glutathione Transferases Examined with Time-resolved Fluorescence ......................25 A. Introduction .......................................................................................................................... 25 B. Theory of Time-resolved Fluorescence Distribution Analysis ............................................ 28 C. Materials and Methods ......................................................................................................... 33 Protein Expression and Purification...................................................................................... 33 Fluorescence Measurement and Analysis ............................................................................. 35 D. Results and Discussion......................................................................................................... 36 Steady-state Fluorescence ..................................................................................................... 36 Time-resolved Fluorescence ................................................................................................. 37 Chapter 3: Calorimetric Analysis of Glutathione Transferases .....................................................46 A. Introduction .......................................................................................................................... 46 B. Differential Scanning Calorimetry and the Variable-barrier Model .................................... 47 C. Materials and Methods ......................................................................................................... 53 Protein Expression and Purification...................................................................................... 53 Differential Scanning Calorimetry (DSC) ............................................................................ 54 Circular Dichroism (CD) ...................................................................................................... 55 D. Results and Discussion......................................................................................................... 55 Differential Scanning Calorimetry ........................................................................................ 55 Circular Dichroism................................................................................................................ 60 Variable-barrier Free Energy Analysis ................................................................................. 63 Chapter 4: Catalytic Promiscuity of GSTs ....................................................................................69 A. Introduction .......................................................................................................................... 69 B. Materials and Methods ......................................................................................................... 70 Enzyme Activity Assays ....................................................................................................... 70 i Promiscuity Quantification ................................................................................................... 72 C. Results and Discussion ......................................................................................................... 72 Glutathione Steady State Kinetic Parameters ....................................................................... 72 Substrate Promiscuity Quantification ................................................................................... 75 Chapter 5: Linking Catalytic Promiscuity and Conformational Heterogeneity ............................78 A. Introduction .......................................................................................................................... 78 B. Results and Discussion ......................................................................................................... 79 C. Conclusions .......................................................................................................................... 82 Glossary .........................................................................................................................................84 References ......................................................................................................................................89 Appendix A: Data Analysis Code ................................................................................................103 Octave Script for Variable-barrier Analysis ....................................................................... 103 Python Script for Promiscuity Calculations ........................................................................ 105 Appendix B: PubChem Substructure Fingerprints ......................................................................107 ii List of Figures Page Figure 1.1 Induced fit schematic ..................................................................................................... 3 Figure 1.2 General conformational selection scheme ..................................................................... 4 Figure 1.3 Molecular structure of glutathione .............................................................................. 15 Figure 1.4 General glutathione transferase catalyzed reaction ..................................................... 16 Figure 1.5 Crystal structures of GSTA1-1 dimer with or monomer ............................................. 19 Figure 1.6 Aligned crystal structures
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