Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis.

摘要: Glutathione S-transferase (GST) was purified from Escherichia coli K-12, and its N-terminal sequence determined to be MKLFYKPGAXSLAS. The gene encoding this cloned mapped at 1731-1732 kilobases on the E. map. It encoded a polypeptide of 201 amino acid residues with calculated molecular weight 22,860. overexpressed product confirmed have GST activity toward 1-chloro-2,4-dinitrobenzene ethacrynic GSH-dependent peroxidase cumene hydroperoxide. relative mass 40,000 by gel chromatography 25,000 SDS-polyacrylamide electrophoresis, indicating homodimeric structure. deduced 54% identical that Proteus mirabilis GST. Although homologies between GSTs mammals were low, many assigned important for enzymatic function or structure in mammalian cytosolic found conserved Therefore, is considered diverged same ancestor other GSTs. A specific tyrosyl residue vicinity N terminus all known has been shown as catalytic alpha, mu, pi class mammals. Tyr5 appeared counterpart residue, replacement phenylalanine did not significantly affect activity. apparently essential