In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage.
Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (IndS) and here called IndS, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these IndS mutant proteins, and of several double mutant proteins containing an IndS mutation and one of several mutations, termed Ind-, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the IndS mutant proteins had a higher maximum rate and a lower apparent pKa than wild-type LexA. Second, the IndS mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of Kcat/Km was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind- mutant protein. We interpret these data to mean that the IndS mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.
Roland
K L
KL
Department of Biochemistry, University of Arizona, Tucson 85721.
Smith
M H
MH
Rupley
J A
JA
Little
J W
JW
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D007531:Isoflurophate; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein