The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases.
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' Tyr(9) by phenylalanine leads to a marked 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-terminal 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 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.
Allardyce
C S
CS
Centre for Mechanisms of Human Toxicity, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.
McDonagh
P D
PD
Lian
L Y
LY
Wolf
C R
CR
Roberts
G C
GC
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D004137:Dinitrochlorobenzene / Q000493:pharmacokinetics; D004976:Ethacrynic Acid / Q000493:pharmacokinetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine
D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D005982:Glutathione Transferase; D004976:Ethacrynic Acid