title: Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase.
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 m) 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]0.5, N-U, at 3.07 M equivalent to free energy change ([Formula: see text]) 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 [Formula: see text] 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.
authors.affiliation: Department of Applied Chemistry, National Chiayi University, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.forename: Yi-Yu
authors.initials: YY
authors.lastname: Chen
authors.forename: Huei-Fen
authors.initials: HF
authors.lastname: Lo
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
keywords: Autocatalytic processing; Bacillus licheniformis; Glutamate; Site-directed mutagenesis; γ-Glutamyltranspeptidase