Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.
LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D009154:Mutation; D010957:Plasmids; D011938:Rec A Recombinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012694:Serine; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D008239:Lysine; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012694:Serine; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein