Functional analysis of the gamma-glutamylcysteine synthetase of Escherichia coli B: effect of substitution of His-150 to Ala.
A high expression system of the gamma-glutamylcysteine synthetase gene (gshl) of Escherichia coli B was constructed, and rapid purification of GSH-I was performed. The active site of GSH-I was analysed by chemical modification, and Lys, Arg and His residues seemed to be involved in the active site of the enzyme. Among them, His residues were substituted to Ala by site-directed mutagenesis, and His-150 was found to be essential for the activity of GSH-I.
Inoue
Y
Y
Research Institute for Food Science, Kyoto University, Japan.
Iba
Y
Y
Yano
H
H
Murata
K
K
Kimura
A
A
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids
D004269:DNA, Bacterial; D006639:Histidine; D005721:Glutamate-Cysteine Ligase; D000409:Alanine