Mutational Analysis of a Highly Conserved PLSSMXP Sequence in the Small Subunit of γ-Glutamyltranspeptidase.

A highly conserved PLSSMXP sequence in the small subunit (S-subunit) of an industrially important γ-glutamyltranspeptidase (GGT) was identified by sequence alignment. Molecular structures of the precursor mimic and the mature form of GGT clearly reveal that this peptide sequence is in close spatial proximity to the self-processing and catalytic sites of the enzyme. To probe the role of this conserved sequence, ten mutant enzymes of GGT were created through a series of deletion and alanine-scanning mutagenesis. SDS-PAGE and densitometric analyses showed that the intrinsic ability of GGT to undergo autocatalytic processing was detrimentally affected by the deletion-associated mutations. However, loss of self-activating capacity was not obviously observed in most of the Ala-replacement mutants. The Ala-replacement mutants had a specific activity comparable to or greater than that of the wild-type enzyme; conversely, all deletion mutants completely lost their enzymatic activity. As compared with GGT, S460A and S461S showed greatly enhanced / values by 2.73- and 2.67-fold, respectively. The intrinsic tryptophan fluorescence and circular dichroism spectral profiles of Ala-replacement and deletion mutants were typically similar to those of GGT. However, heat and guanidine hydrochloride-induced unfolding transitions of the deletion-associated mutant proteins were severely reduced as compared with the wild-type enzyme. The predictive mutant models suggest that the microenvironments required for both self-activation and catalytic reaction of GGT can be altered upon mutations.