USE OF SITE-DIRECTED CHEMICAL MODIFICATION TO STUDY AN ESSENTIAL LYSINE IN ESCHERICHIA-COLI LEADER PEPTIDASE

Escherichia coli leader peptidase, which catalyzes the cleavage of sig nal peptides from pre-proteins, is an essential, integral membrane ser ine peptidase that has its active site residing in the periplasmic spa ce. It contains a conserved lysine residue that has been proposed to a ct as the general base, abstracting the proton from the side chain hyd roxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidas e, we have combined site-directed mutagenesis and chemical modificatio n methods to introduce unnatural amino acid side chains at the 145-pos ition. We show that partial activity can be restored to an inactive K1 45C leader peptidase mutant by reacting it with 2-bromoethylamine . HB r to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine . HBr and 2-mercapto ethylamine also allowed for partial restoration of activity showing th at there is some flexibility in the length requirements of this essent ial residue. Modification with (2-bromoethyl)trimethylammonium . Br to form a positively charged, nontitratable side chain at the 145-positi on failed to restore activity to the inactive K145C leader peptidase m utant. This result, along with an inactive K145R mutant result, suppor ts the claim that the lysine side chain at the 145-position is essenti al due to its ability to form a hydrogen bond(s) or to act as a genera l base rather than because of an ability to form a critical salt bridg e. We find that leader peptidase processes the pre-protein substrate, pro OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent p K(a) values for titratable groups at approximately 8.7 and 9.3 are rev ealed. We show that the lysine modifier maleic anhydride inhibits lead er peptidase by reacting with lysine 145. The results of this study ar e consistent with the hypothesis that the lysine at the 145-position o f leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based an th e structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.