Effect of Site-Directed Mutations on Processing and Activity of γ-Glutamyltranspeptidase of Escherichia coli K-12

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  • HASHIMOTO Wataru
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • SUZUKI Hideyuki
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • YAMAMOTO Kenji
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University
  • KUMAGAI Hidehiko
    Department of Food Science and Technology, Faculty of Agriculture, Kyoto University

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γ-Glutamyltranspeptidase [EC 2. 3. 2. 2] of Escherichia coli K-12 is thought to be synthe-sized from a single precursor polypeptide into a heterodimeric form through post-translational processing. Cells of a γ-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 γ-glutamyltranspeptidase, membrane fraction only contained the precursor of γ-glutamyltranspeptidase, and no precursor of γ-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 γ-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 γ-glutamyltranspeptidase and this processing is essential to the expression of γ-glutamyltranspeptidase activity of E. coli K-12.

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