PMID:8537328
| Citation |
Hashimoto, W, Suzuki, H, Yamamoto, K and Kumagai, H (1995) Effect of site-directed mutations on processing and activity of gamma-glutamyltranspeptidase of Escherichia coli K-12. J. Biochem. 118:75-80 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Escherichia coli/enzymology; Escherichia coli/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phenotype; Protein Processing, Post-Translational; gamma-Glutamyltransferase/genetics; gamma-Glutamyltransferase/metabolism |
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