The Effects of Mutations in the Carboxyl-Terminal Region on the Catalytic Activity of Escherichia coli Signal Peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp 300 and Arg 77 , Arg 222 , Arg 315 and Arg 318 are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His 323 was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly 321 -His 323 or Ile 319 -His 323 as well as the point mutation of Ile 322 to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile 322 to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.