Amino acid residue 104 in an alpha-class glutathione S-transferase is essential for the high selectivity and specificity of the enzyme for 4-hydroxynonenal.
Murine mGSTA4-4 is a glutathione S-transferase with high activity and specificity for products of lipid peroxidation, including the cytotoxic 4-hydroxynonenal (4-HNE). Physiological relevance of this enzyme in the defense against effects of oxidative stress can be inferred from the above biochemical properties, and has been also directly demonstrated by us in vivo. The identification of residues responsible for the high activity toward 4-HNE is facilitated by the availability of X-ray crystal structures of mGSTA4-4 and of hGSTA1-1, a structurally related enzyme which lacks activity for 4-HNE. Residues likely to be involved in 4-HNE recognition were identified by molecular modeling. One such residue, M104, was mutated to E104, as present in hGSTA1-1. The resulting M104E mutant had unchanged catalytic properties toward the model substrate 1-chloro-2,4-dinitrobenzene. However, the Km of mGSTA4-4(M104E) for 4-HNE was increased more than sevenfold, while the Vmax for that substrate remained essentially unchanged. We conclude that M104 codetermines the recognition and binding of 4-HNE to the active center of mGSTA4-4.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, and VA John McClellan Memorial Hospital, Little Rock 72205, USA.
B
B
Nanduri
J B
JB
Hayden
Y C
YC
Awasthi
P
P
Zimniak
D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008054:Lipid Peroxides / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000447:Aldehydes; D004137:Dinitrochlorobenzene; D008054:Lipid Peroxides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C027576:4-hydroxy-2-nonenal