The role of tyrosine-9 and the C-terminal helix in the catalytic mechanismof Alpha-class glutathione S-transferases

Glutathione S-transferases (GSTs) play a key role in the: metabolism of dru gs and xenobiotics. To investigate the catalytic mechanism, substrate bindi ng and catalysis by the wild-type and two mutants of GST A1-1 have been stu died. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a ma rked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-termi nal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The bindin g 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 substr ate with the small thiols 2-mercaptoethanol and dithiothreitol. This sugges ts that GSH binding produces a conformational change which is transmitted t o the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (' turnover nu mber') for conjugation, perhaps by increasing the proportion of the substra te bound productively. Neither of these two effects of GSH analogues are se en in the C-terminally truncated enzyme, indicating a role for the C-termin al helix in the GSH-induced conformational change.