title: A serine and a lysine residue implicated in the catalytic mechanism of the Escherichia coli leader peptidase.
abstract: We report that a thiol leader peptidase, produced by replacing the critical serine at position 90 with a cysteine residue, is enzymatically active. In contrast to the wild-type leader peptidase, the thiol enzyme can be inactivated with N-ethylmaleimide, a cysteine-specific reagent. This strongly suggests that the serine 90 is involved in catalysis and is located at the active site. Of the three conserved basic residues in the signal peptidase family, only lysine 145 appears to be critical for catalysis; when lysine 145 was mutated to an alanine residue, leader peptidase K145A protein was inactive both in vitro and in vivo. A control experiment showed that the K145A mutant competes with the wild-type leader peptidase for substrate binding, confirming that the K145A mutation did not cause a global conformational change. The data provides evidence that catalysis of leader peptidase is carried out by a serine-lysine dyad.
authors.affiliation: Department of Chemistry, Ohio State University, Columbus 43210.
authors.forename: W R
authors.initials: WR
authors.lastname: Tschantz
authors.forename: M
authors.initials: M
authors.lastname: Sung
authors.forename: V M
authors.initials: VM
authors.lastname: Delgado-Partin
authors.forename: R E
authors.initials: RE
authors.lastname: Dalbey
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism*; D008565:Membrane Proteins; D009154:Mutation; D012694:Serine / Q000378:metabolism*; D012697:Serine Endopeptidases
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D012694:Serine; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D008239:Lysine; D005033:Ethylmaleimide