Identification of Tyrosine 79 in the Tocopherol Binding Site of Glutathione S-Transferase Pi†
Abstract
Alpha-tocopherol, the most abundant form of vitamin E present in humans, is a noncompetitive inhibitor of glutathione S-transferase pi (GST pi), but its binding site had not been located. Tocopherol iodoacetate (TIA), a reactive analogue, produces a time-dependent inactivation of GST pi to a limit of 25% residual activity. The rate constant for inactivation, kobs, exhibits a nonlinear dependence on reagent concentration, with KI = 19 μM and kmax = 0.158 min-1. Complete protection against inactivation is provided by tocopherol and tocopherol acetate, whereas glutathione derivatives, electrophilic substrate analogues, buffers, or nonsubstrate hydrophobic ligands have little effect on kobs. These results indicate that TIA reacts as an affinity label of a distinguishable tocopherol binding site. Loss of activity occurs concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. Isolation of the labeled peptide from the tryptic digest shows that Tyr79 is the only enzymic amino acid modified. The Y79F, Y79S, and Y79A mutant enzymes were generated, expressed, and purified. Changing Tyr79 to Ser or Ala, but not Phe, renders the enzyme insensitive to inhibition by either tocopherol or tocopherol acetate as demonstrated by increases of at least 49-fold in KI values as compared to the wild-type enzyme. These results and examination of the crystal structure of GST pi suggest that tocopherols bind at a novel site, where an aromatic residue at position 79 is essential for binding.
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This work was supported by NIH Grant R01-CA66561 (to R.F.C.) and by NIH Fellowship 1F31 GM75387 (to L.A.R.).
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