Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1

Abstract

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

Molecular Cloning, Characterization, and Expression in Escherichia coli of Full-length cDNAs of Three Human Glutathione S-Transferase Pi Gene Variants

“…These results are similar to those in a previous report in which a recombinant GSTP1b-1b protein expressed from a cDNA obtained artificially by site-directed mutagenesis of hGSTP1*A, was shown to have a 4-fold higher K m (CDNB) than the hGSTP1*A encoded protein (41). At high substrate (CDNB) concentration, V max of the reaction catalyzed by all three GST Pi proteins differed only modestly and was in the range reported in other studies of both tissue and recombinant GST Pi proteins (41,(45)(46)(47). K cat for CDNB was also similar for all three proteins, thus resulting in utilization ratios, K cat /K m , for GSTP1b-1c and GSTP1c-1c that were 3.3-and 4-fold lower, respectively, than that of GSTP1a-1a.…”

“…Every 15 min, over 1 h, residual GST activity was determined as described in the text using CDNB as substrate. 113 with Val in the x-ray crystallographic structure of the human placental GSTP1a, co-crystallized with S-hexylglutathione (28,45). The resulting three-dimensional structures were energy minimized.…”

“…Structural Analysis of Variant GST Pi Proteins-Data from the previously reported x-ray crystallographic structure of GSTP1a (28,45) have shown the key amino acid residues lining the putative H-site of human placental GST Pi to consist of Tyr 7 , Tyr 108 , Val 10 , Val 35 , Phe 8 , and Gly 205 . We modeled the effects of the amino acid substitutions in the variant GST Pi proteins on the structure of the resulting peptides, particularly in the H-site pocket.…”

“…K m and V max for CDNB were determined over a concentration of 0 -5 mM CDNB and 2.5 mM GSH. Ile 104 and both Ile 104 and Ala 113 with Val, respectively, in the x-ray crystallographic structure of the human placental GSTP1a (28,45), imported from the Brookhaven Protein Data Bank. The structural modeling was performed using the SYBYL molecular modeling program.…”

Epitope mapping of a monoclonal antibody to human glutathione transferase P1–1 the binding of which is inhibited by glutathione

“…Since the involvement of GSTs in drug resistance was first suggested, the three-dimensional structures of these enzymes crystallized with GSH or its analogues have been extensively studied to aid development of specific inhibitors [9][10][11][12]. Several amino acid residues have been identified, the replacement of which results in marked decreases in activity or alterations in the K m values for GSH [13][14][15][16]. Most residues constituting the G-site are localized in the N-terminal half of the subunit (domain I) [9,11].…”