Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model.
The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.
Department of Biochemistry, University of Arizona, Tucson 85721.
K L
KL
Roland
J W
JW
Little
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007531:Isoflurophate / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D003488:Cyanogen Bromide; D007531:Isoflurophate; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein