Sequence comparison showed that residues Thr407, Asp433, and Met464 in the small subunit of Escherichia coli gamma-glutamyltranspeptidase (EcGGT) were conserved in the aligned enzymes. In this study, we further investigated the functional significance of these conserved residues by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 cells and purified to near homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown no GGT activity under enzyme assay conditions and activity staining. Furthermore, mutations on these residues impaired the capability of autocatalytic processing of the enzyme. Based on these observations, it is concluded that these residues play an important role in the enzyme maturation.