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J. Biol. Chem., Vol. 282, Issue 32, 23264-23274, August 10, 2007

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

From the Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610

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.

Received for publication, January 30, 2007 , and in revised form, June 7, 2007.

^* This work was supported by a Drug Metabolism, Pharmacokinetics, and Toxicology Postdoctoral Fellowship from the University of Washington School of Pharmacy (to L. H.) and by National Institutes of Health Grant T32 GM07750 (to L. S. and M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5 and Tables S1 and S2.

¹ To whom correspondence should be addressed: Box 357610, Dept. of Medicinal Chemistry, University of Washington, Seattle, WA 98195-7610. Tel.: 206-685-0379; Fax: 206-685-3252; E-mail: winky{at}u.washington.edu.

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