The high activity of rat glutathione transferase 8-8 with alkene substrates is dependent on a glycine residue in the active site.
Rat glutathione transferase (GST) 8-8 displays high catalytic activity with alpha, beta-unsaturated carbonyl compounds, including lipid peroxidation products such as 4-hydroxyalkenals. The catalytic efficiency of the related class Alpha GST 1-1 is substantially lower with the same substrates. Chimeric enzymes were prepared by replacing N-terminal subunit 8 segments of different lengths (6, 25, or 100 residues) with corresponding sequences from subunit 1 using recombinant DNA techniques. The chimeric subunit r1(25)r8, containing 25 amino acid residues from subunit 1, had the same low activity with alkenal substrates as that displayed by subunit 1. Mutation of Ala-12 into Gly in r1(25)r8 gave rise to the high alkenal activity characteristic of subunit 8, showing the importance of amino acid residue 12 for the activity. However, other structural determinants are also essential, as demonstrated by the corresponding Ala-12-->Gly mutation in subunit 1, which did not afford high alkenal activity. The results show that a single point mutation in a GST subunit may give rise to a 100-fold increase in catalytic efficiency with certain substrates. Introduction of such mutations may have contributed to the biological evolution of GST isoenzymes with altered substrate specificities and may also find use in the engineering of GSTs for novel functions.
Department of Biochemistry, Uppsala University, Sweden.
R
R
Björnestedt
S
S
Tardioli
B
B
Mannervik
D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D017931:DNA Primers; D005091:Exons; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005998:Glycine; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000475:Alkenes; D017931:DNA Primers; D007527:Isoenzymes; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D005998:Glycine