Effect of site-directed mutations on processing and activity of gamma-glutamyltranspeptidase of Escherichia coli K-12.
gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 is thought to be synthesized from a single precursor polypeptide into a heterodimeric form through post-translational processing. Cells of a gamma-glutamyltranspeptidase-overproducing transformant of E. coli K-12 were fractionated and the localization of the enzyme was examined by Western blot analysis. The periplasmic fraction only contained the mature form of gamma-glutamyltranspeptidase, membrane fraction only contained the precursor of gamma-glutamyltranspeptidase, and no precursor of gamma-glutamyltranspeptidase was detected in the cytoplasmic fraction. Amino acid residues at the cleavage site for processing into the large and small subunits were substituted by site-directed mutagenesis. The processing phenotypes of six mutants were examined by Western blot analysis, and their gamma-glutamyltranspeptidase activities were measured. Mutations at the N-terminal amino acid residues of the small subunit (Thr-391, Thr-392, and His-393) prevented the maturation of the enzyme and the immature mutants exhibited no enzymatic activity. A mutation at the C-terminal residue of the large subunit (Gln-390) had less effect on the processing and enzymatic activity. These results suggest that the sequence of threonyl-threonyl-histidinyl residues at the N-terminal of the small subunit is very important for the processing of E. coli K-12 gamma-glutamyltranspeptidase and this processing is essential to the expression of gamma-glutamyltranspeptidase activity of E. coli K-12.
Hashimoto
W
W
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Suzuki
H
H
Yamamoto
K
K
Kumagai
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000378:metabolism
D005723:gamma-Glutamyltransferase