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J Biol Chem. 1997 Apr 11;272(15):9994-10003.

Use of site-directed chemical modification to study an essential lysine in Escherichia coli leader peptidase.

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Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.


Escherichia coli leader peptidase, which catalyzes the cleavage of signal peptides from pre-proteins, is an essential, integral membrane serine peptidase that has its active site residing in the periplasmic space. It contains a conserved lysine residue that has been proposed to act as the general base, abstracting the proton from the side chain hydroxyl group of the nucleophilic serine 90. To help elucidate the role of the essential lysine 145 in the activity of E. coli leader peptidase, we have combined site-directed mutagenesis and chemical modification methods to introduce unnatural amino acid side chains at the 145-position. We show that partial activity can be restored to an inactive K145C leader peptidase mutant by reacting it with 2-bromoethylamine.HBr to produce a lysine analog (gamma-thia-lysine) at the 145-position. Modification with the reagents 3-bromopropylamine.HBr and 2-mercaptoethylamine also allowed for partial restoration of activity showing that there is some flexibility in the length requirements of this essential residue. Modification with (2-bromoethyl)trimethylammonium.Br to form a positively charged, nontitratable side chain at the 145-position failed to restore activity to the inactive K145C leader peptidase mutant. This result, along with an inactive K145R mutant result, supports the claim that the lysine side chain at the 145-position is essential due to its ability to form a hydrogen bond(s) or to act as a general base rather than because of an ability to form a critical salt bridge. We find that leader peptidase processes the pre-protein substrate, pro-OmpA nuclease A, with maximum efficiency at pH 9.0, and apparent pKa values for titratable groups at approximately 8.7 and 9.3 are revealed. We show that the lysine modifier maleic anhydride inhibits leader peptidase by reacting with lysine 145. The results of this study are consistent with the hypothesis that the lysine at the 145-position of leader peptidase functions as the active site general base. A model of the active site region of leader peptidase is presented based on the structure of the E. coli UmuD', and a mechanism for bacterial leader peptidase is proposed.

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