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Contribution of the mu loop to the structure and function of rat glutathione transferase M1-1

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA

(RECEIVED February 1, 2006; FINAL REVISION February 24, 2006; ACCEPTED February 24, 2006)

The "mu loop", an 11-residue loop spanning amino acid residues 33–43, is a characteristic structural feature of the mu class of glutathione transferases. To assess the contribution of the mu loop to the structure and function of rat GST M1-1, amino acid residues 35–44 (³⁵GDAPDYDRSQ⁴⁴) were excised by deletion mutagenesis, resulting in the "Deletion Enzyme." Kinetic studies reveal that the K_m values of the Deletion Enzyme are markedly increased compared with those of the wild-type enzyme: 32-fold for 1-chloro-2,4-dinitrobenzene, 99-fold for glutathione, and 880-fold for monobromobimane, while the V_max value for each substrate is increased only modestly. Results from experiments probing the structure of the Deletion Enzyme, in comparison with that of the wild-type enzyme, suggest that the secondary and quaternary structures have not been appreciably perturbed. Thermostability studies indicate that the Deletion Enzyme is as stable as the wild-type enzyme at 4°C and 10°C, but it rapidly loses activity at 25°C, unlike the wild-type enzyme. In the temperature range of 4°C through 25°C, the loss of activity of the Deletion Enzyme is not the result of a change in its structure, as determined by circular dichroism spectroscopy and sedimentation equilibrium centrifugation. Collectively, these results indicate that the mu loop is not essential for GST M1-1 to maintain its structure nor is it required for the enzyme to retain some catalytic activity. However, it is an important determinant of the enzyme's affinity for its substrates.

Keywords: glutathione S-transferase M1-1; deletion mutagenesis; mu loop; substrate specificity

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062129506.

Abbreviations: GST, glutathione S-transferase; WT, wild-type GST M1-1; Deletion Enzyme, mu loop (amino acid residues 35–44) deletion mutant GST M1-1 enzyme; GST A1-1, alpha class GST; GST P1-1, pi class GST; GSH, glutathione; GSHME, glutathione monoethyl ester; CDNB, 1-chloro-2, 4-dinitrobenzene; mBBr, monobromobimane, 4-bromomethyl-3,6,7-trimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione; Tris, tris(hydroxymethyl)aminomethane; LB, Luria-Bertani; EDTA, disodium ethylenediamine tetraacetate; DMF, N, N'-dimethylformamide; IPTG, isopropyl--D-thiogalactoside; TFA, trifluoroacetic acid; CD, circular dichroism; ESI-MS, electrospray ionization mass spectrometry; NMR, nuclear magnetic resonance; rpm, revolutions per minute.

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