Stereochemical complexities in the glutathione S-transferase catalyzed detoxification of 4-hydroxynonenal

The A-class glutathione S-transferases (GSTs) play an important role in detoxification of oxidative stress products, with the GSTA4-4 isoform distinguished for its high catalytic efficiency with 4-hydroxynonenal (HNE), a toxic electrophile generated endogenously during lipid peroxidation. Previous work on human GSTA4-4 has focused on racemic HNE and the major emphasis of this dissertation is the characterization of substrate stereoselectivity and stereoselectivity of product formation in the human GSTA4-4-catalyzed conjugation reaction.;LC/MS analyses presented in Chapter 2 explicitly demonstrate that GSTA4-4 conjugates HNE with glutathione to generate diastereomeric products in a completely stereoselective manner that is not maintained in the spontaneous reaction of HNE and glutathione. Furthermore, assignment of the stereochemical configurations of the different diastereomers through LC/MS and NMR characterizations is consistent with a GSTA4-4-catalyzed nucleophilic attack that yields only the S-configuration at the site of conjugation, regardless of initial substrate chirality. In order to address substrate stereoselectivity, the kinetic studies discussed in Chapter 3 examine the simultaneous metabolism of the individual HNE enantiomers from within the racemic mixture. In contrast to the strict stereoselectivity of product formation, these results indicate that GSTA4-4 exhibits a remarkably high apparent catalytic efficiency for both enantiomeric substrates, with only a modest preference for S -HNE in the presence of both enantiomers.;The final chapter focuses on the stereoselectivity of GSTA4-4 as compared with other GST isoforms. In addition to LC/MS comparisons, the GSTA1-1 GIMFhelix mutant crystal structures detailed in Chapter 4 provide structural information that indicates conformational heterogeneity can be inversely correlated with stereoselectivity in A-class GSTs. Collectively, the results establish that an aromatic-aromatic interaction between Phe111 and Tyr217, as well as a proline residue at position 10, is necessary to attain the observed stereoselectivity within the GSTA4-4 active site. A model for initial HNE-binding is also proposed, which illustrates how Arg15 could be exploited in order to achieve low substrate stereoselectivity in combination with the strict stereoselectivity of product formation, thereby allowing for the detoxification of both endogenous enantiomers while producing only a select set of diastereomers with potential stereochemical implications concerning their fates in biological tissues.