Journal
FEBS OPEN BIO
Volume 2, Issue -, Pages 298-304Publisher
WILEY
DOI: 10.1016/j.fob.2012.09.007
Keywords
Bacillus licheniformis; gamma-Glutamyltranspeptidase; Glutamate; Site-directed mutagenesis; Autocatalytic processing
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Funding
- National Science Council of Taiwan [NSC 97-2628-B-415-001-MY3, NSC 100-2313-B-415-003-MY3]
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The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis gamma-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-m) of 47.7-69.4 degrees 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 similar to 1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl](0.5, N-U), at 3.07 M equivalent to free energy change (Delta G(N-U)(H2O)) of 14.53 kcal/mol for the N -> U process, whereas the denaturation midpoints for the mutant enzymes were 1.31-2.99Mequivalent to Delta G(N-U)(H2O) 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. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
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