Functional Promiscuity Correlates with Conformational Heterogeneity in A-class Glutathione S-Transferases

Abstract

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.

A Quantitative Index of Substrate Promiscuity

“…Several biophysical techniques (32) indicate that A1-1 is also more structurally dynamic than A4-4, suggestive of a correlation between promiscuity and increased dynamic flexibility (and possibly decreased stability). The promiscuity index provides a quantitative basis for this comparison, which previously we could only describe "intuitively".…”

“…Because I and J indices are independent of an enzyme's overall level of activity, they allow us to address the open question (4,6,9,14,20,23,32) of whether functional promiscuity can be achieved simultaneously with catalytic efficiency. If substrate specificity is correlated with catalytic efficiency, then a strong negative correlation between promiscuity and the catalytic efficiency for the most-favored substrate would be anticipated.…”

“…There is currently no quantitative measure of an enzyme's substrate promiscuity, which makes it impossible to compare the promiscuous behavior of different proteins or to study correlations between promiscuity and catalytic efficiency or protein stability. For example, in our own work with GSTs it has been impossible to consider quantitatively how promiscuity of a series of mutants is related to their dynamic properties (32).…”

Ensemble Perspective for Catalytic Promiscuity

“…However, in contrast to the well established contribution of transition state stabilization to catalysis and specificity, the molecular bases for biological promiscuity are unknown. For a few specific examples, x-ray crystallographic or spectroscopic methods have suggested structural or dynamic mechanisms by which enzymes achieve catalytic promiscuity (4,12,13,21). In general, promiscuity has been correlated with increased structural "flexibility" of enzymes, although there has been no description of the detailed relationship between motion on different time scales and functional promiscuity (3,12,19,22).…”

“…It is now clear that functionally useful promiscuity plays a role in the immune system (7)(8)(9)(10), in signal transduction pathways (11), and in enzymatic detoxification (12)(13)(14)(15). In these cases, the promiscuous interaction of a single enzyme or protein with multiple substrates or partners represents an evolutionary end point; the optimal function of the protein includes substrate promiscuity.…”