Contribution of amino acid residue 208 in the hydrophobic binding site to the catalytic mechanism of human glutathione transferase A1-1

Abstract

Glutathione transferases (GSTs) catalyze the nucleophilic attack of the thiolate of glutathione on a variety of noxious, often hydrophobic, electrophiles. The interactions responsible for the binding of glutathione have been deduced in great detail from the 3-dimensional structures that have been solved for three different GSTs, each a member of a distinct structural class. However, the interactions of the electrophilic substrates with these enzymes are still largely unexplored. The contribution of the active-site Met208 to aromatic and benzylic chloride substitution reactions catalyzed by human class Alpha GST A1-1 has been evaluated by comparison of wild-type enzyme with variants mutated in position 208. The results show that the amino acid residue at position 208 primarily affects the aromatic substitution reaction, tested with 1-chloro-2,4-dinitrobenzene as substrate, possibly by interacting with the delocalized negative charge of the substituted ring structure in the transition state.

A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases

“…This is consistent with the binding of GSH not being significantly influenced by the Cterminal region (12,16,17,52,53).…”

“…It would appear that the reduction in catalytic efficiency is not due to an increase in K m CDNB but rather a decrease in k cat CDNB (not determined). A decrease in k cat CDNB , in turn, would not be due to a decreased rate of product release, because mobility-enhancing perturbations in the Cterminal region generally increase the rate of product release (12,51,53). Rather, it is most likely that the affinity of the H-site for CDNB is reduced, which is consistent with the link between productive binding of CDNB and the dynamics of the C-terminal region (9).…”

“…The C-terminal region of GST A1-1 plays an important role in guiding the substrates into the transition state (13). The diminished stabilization effect observed for the D209G mutant is, however, not as pronounced as that observed when Met-208, the hydrophobic staple motif at the N terminus of helix 9, is altered (53). It would appear that the reduction in catalytic efficiency is not due to an increase in K m CDNB but rather a decrease in k cat CDNB (not determined).…”

Functional Studies of Tyrosine 108 Residue in the Active Site of Human Glutathione S-Transferase P1-1

“…44 X-ray crystallographic studies have also suggested that Tyr 108 in hGST P1-1 is located at the substrate-binding site and it is in a structurally conserved position within the mu, pi and theta classes, and appears to be one of the few polar residues contributing to the H-site. [27][28][29][30][31][32] In order to elucidate the precise enzyme-substrate interactions responsible for the catalytic properties of GST, the mutant enzymes of Tyr 108 residue in hGST P1-1 were expressed in E. coli, purified to electrophoretic homogeneity by affinity column chromatography and examined the enzymatic properties of the mutated enzymes. The substitutions of Tyr 108 with alanine, phenylalanine and tryptophan greatly affected the catalytic properties of hGST P1-1 toward a number of substrates, as discussed below.…”

“…Subsequent crystallographic studies of all five GST classes show that the H-site is quite different among them and very little is known about the key determinants of xenobiotic substrate specificity. 10,11,[24][25][26][27] Only a few amino acid residues have been identified as key determinants of the H-site : Tyr 115 in rat mu GST (isoenzymes 3-3 and 4-4), 28,29 Met 208 in GST A1-1, 30 Ile 104 in GST P1-1, 31 and Val 10, Arg 11, and Val 104 in the murine class pi GST. 32 Previously, we reported the importance of Tyr 108 for the binding of the electrophilic substrate of hGST P1-1.…”