Structural Dynamics and Reaction Reversibility of Specific vs. Promiscuous Detoxification Enzymes

Abstract

Detoxification enzymes occupy a unique niche in biology. Unlike most enzymes with defined roles in biochemical pathways, detoxification enzymes have evolved to bind and metabolize many chemically unrelated structures ¬– to be promiscuous – rather than to optimize catalysis towards a preferred substrate or set of substrates. It is likely that detoxification enzymes have unique properties that allow them to achieve their remarkable promiscuity. This thesis focuses on understanding some of these properties by contrasting the behavior of two highly promiscuous detoxification enzymes cytochrome P450 3A4 (CYP3A4) and glutathione transferase A1-1 (GSTA1-1) with that of structurally related, substrate-specific counterparts CYP19A1 and GSTA4-4. Two aspects of enzyme behavior are considered: (I) structural dynamics in the absence and presence of ligands, and (II) reversibility of catalysis.Chapters 2 and 3 describe the substrate-dependent structural dynamics of membrane-embedded CYP3A4 and CYP19A1, respectively, via hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations. Comparison of the structural dynamics of CYP3A4 bound to eight ligands reveals subtle and ligand-dependent effects on dynamics that are primarily distributed in the flexible regions of the enzyme. No correlation with known ligand properties such as size, binding affinity, and CYP3A4 allosteric function is observed. The dynamic signatures of the two azole-based inhibitors are distinct from the rest of the examined ligands, though a correlation with ligand type needs to be further explored. The largest effects on dynamics are found in the important F-G region and the most typical effect is an increase in dynamics of the F-helix and connecting loops, suggesting a counterintuitive increase in flexibility when CYP3A4 is bound to ligands in a membrane environment. By contrast, the native steroidal ligand of CYP19A1 causes a clear suppression of dynamics that is most pronounced at the substrate binding site, protein-membrane interface, and access channel. Suppression of dynamics is smaller with two nonsteroidal ligands, particularly at the membrane interface and access channel. The discrimination between the dynamic signatures of CYP19A1 bound to native and nonnative ligands contrasts with the ambiguous small effects observed with CYP3A4 ligands. Collectively, the results are consistent with the substrate selectivity profiles of the two isoforms. Chapter 4 explores the reversibility of glutathione conjugation to a lipid alkenal substrate by two closely related GST isoforms, GSTA1-1 and GSTA4-4, via 1H NMR, UV-VIS spectroscopy, and LC-MS. Unlike its promiscuous counterpart, GSTA4-4 more readily catalyzes the conjugation reaction of trans-2-nonenal in the reverse direction, introducing a novel differentiating aspect in the catalytic mechanisms of the two isoforms. A speculative implication of the chapter is whether the reaction reversibility of GSTA4-4 contributes to regulation, in addition to detoxification, of endogenous lipid alkenal products.