Please use this identifier to cite or link to this item: http://nopr.niscair.res.in/handle/123456789/5795
Title: Contribution of Ser463 residue to the enzymatic and autoprocessing activities of Escherichia coli -glutamyltranspeptidase
Authors: Hsu, Wen-Hwei
Ong, Ping-Lin
Chen, Shih-Chun
Lin, Long-Liu
Keywords: E coli;-Glutamyltranspeptidase;Site-specific mutagenesis;Autocatalytic processing;Tryptophan emission fluorescence;Circular dichroism
Issue Date: Aug-2009
Publisher: CSIR
Abstract:  A serine residue Ser463, required for proper function of E. coli -glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into - and -subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.
Page(s): 281-288
URI: http://hdl.handle.net/123456789/5795
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.46(4) [August 2009]

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