Chemical modification of a cloned glutathione S-transferase from Schistosoma japonicum: evidence for an essential histidine residue.
Diethylpyrocarbonate (DEP) inhibits the catalytic activity of a cloned glutathione S-transferase from Schistosoma japonicum (Sj26GST) with a second-order rate constant of 474 M-1 min-1 at pH 7.0 and 25 degrees C. There is an accompanying increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. There was no evidence that tyrosine or cysteine residues were modified by DEP treatment nor did the enzyme undergo any major conformational change. Activity can be restored by treating the DEP-modified enzyme with hydroxylamine and the pH curve for inactivation indicates involvement of a residue with a pKa of 7.3. Complete inactivation of Sj26GST requires the modification of six histidine residues per subunit. Statistical analysis of residual enzyme activity versus number of groups modified showed that of the six modifiable groups, only one is critical for activity. Substrate protection suggests that this essential histidine residue is at or near the active site.
Walker
J
J
Institute of Biological Sciences, University of Wales, Aberystwyth, Penglais, United Kingdom.
Crowley
P
P
Barrett
J
J
D000818:Animals; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D005982:Glutathione Transferase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics
D006898:Hydroxylamines; D011994:Recombinant Proteins; D019811:Hydroxylamine; D006639:Histidine; D005982:Glutathione Transferase; D005978:Glutathione; D004047:Diethyl Pyrocarbonate; C033550:hexylglutathione