Site-directed mutagenesis and chemical modification of histidine residues on an alpha-class chick liver glutathione S-transferase CL 3-3. Histidines are not needed for the activity of the enzyme and diethylpyrocarbonate modifies both histidine and lysine residues.

Abstract

Each chick liver glutathione S-transferase CL 3 subunit contains three histidine residues: His142, His158 and His228. CL 3-3 can be inactivated by treating with diethylpyrocarbonate. The inactivation process is pH dependent and the pKa of the modified residue is 6.4. The second-order inhibition rate constant is 741 M-1min-1 at pH 7.0. Based on difference-spectrum and kinetic analysis, inactivation coincides with the modification of one histidine residue. However, hydroxylamine treatment of the diethylpyrocarbonate-modified enzyme only partially restored the activity (30-50%) of CL 3-3. By tryptic mapping and amino acid sequence analysis, His228 and Lys14 have been identified as the modified residues. Mutants with histidine to serine replacement (H142S and H158S) or C-terminal histidine deletion (des-H228) were constructed and over-expressed in Spodoptera frugiperda cells using a baculovirus system. The mutants are enzymically active. Furthermore, the des-H228 mutant can be inactivated by diethylpyrocarbonate. These results support the conclusion that histidines are not involved in the enzymic mechanism of CL 3-3.