Abstract
The role of glutamate 398 in the autocatalytic processing of
Bacillus licheniformis
γ‐glutamyltranspeptidase (
Bl
GGT) 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
Bl
GGT. As compared with the wild‐type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of
Bl
GGT, 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
Bl
GGT 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 (
) 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
Bl
GGT.