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Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase.
- Source :
- FEBS Open Bio; Dec2012, Vol. 2, p298-304, 7p
- Publication Year :
- 2012
-
Abstract
- Abstract: The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was explored by site-directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal-affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild-type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two-state unfolding process with a transition point (T <subscript>m</subscript>) of 47.7–69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild-type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ∼1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]<subscript>0.5, N–U</subscript>, at 3.07 M equivalent to free energy change () of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT. [Copyright &y& Elsevier]
Details
- Language :
- English
- ISSN :
- 22115463
- Volume :
- 2
- Database :
- Supplemental Index
- Journal :
- FEBS Open Bio
- Publication Type :
- Academic Journal
- Accession number :
- 84193390
- Full Text :
- https://doi.org/10.1016/j.fob.2012.09.007