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.
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.
Chang
L H
LH
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Tam
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002645:Chickens; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009036:Moths; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D013053:Spectrophotometry; D014357:Trypsin
D006898:Hydroxylamines; D019811:Hydroxylamine; D006639:Histidine; D005982:Glutathione Transferase; D014357:Trypsin; D008239:Lysine; D004047:Diethyl Pyrocarbonate