Abstract
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.
Highlights
Attempts have been made to classify glutathione transferases on the basis of activities with different substrates (Boyland and Chasseaud, 1969), but in general substrate specificities are largely overlapping, and members within the same structural class can diverge very significantly in their substrate preferences
We have previously demonstrated that fully functional chimeric GSTs can be constructed by replacing the C-terminal one-third of a human GST structure with the corresponding segment of a rat GST (Mannervik et al, 1990; Bjornestedt et al, 1992)
In the present investigation it was decided to construct chimeric GSTs from segments of rat GST 8 – 8 and rat GST 1–1 in an attempt to locate amino acid residues essential for the high catalytic activity with alkene substrates
Summary
Attempts have been made to classify glutathione transferases on the basis of activities with different substrates (Boyland and Chasseaud, 1969), but in general substrate specificities are largely overlapping, and members within the same structural class can diverge very significantly in their substrate preferences. Four of the variant amino acids are located at the active site (Sinning et al, 1993) These limited structural differences appear to govern the significant differences in substrate specificities reported (Chow et al, 1988; Burgess et al, 1989). A class Alpha isoenzyme, GST 8 – 8, has been identified, which is characterized by high catalytic activity with 4-hydroxyalkenals (Jensson et al, 1986) and other activated alkenes (Stenberg et al, 1992). In the present investigation it was decided to construct chimeric GSTs from segments of rat GST 8 – 8 and rat GST 1–1 in an attempt to locate amino acid residues essential for the high catalytic activity with alkene substrates. The results of the present investigation show that the characteristic high catalytic efficiency of GST 8 – 8 with activated alkenes is critically dependent on Gly-12 in the active-site region
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
