Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.
The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases
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Claire S. ALLARDYCE, Paul D. MCDONAGH, Lu-Yun LIAN, C. Roland WOLF, Gordon C. K. ROBERTS; The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases. Biochem J 1 November 1999; 343 (3): 525–531. doi: https://doi.org/10.1042/bj3430525
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