Conformational Heterogeneity and Catalytic Promiscuity in Glutathione Transferases
Honaker, Matthew T.
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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.
- Medicinal chemistry