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.Escherichia coli LexA and X cI repressors are inactivated in vivo by a specific cleavage reaction that cuts a conserved Ala-Gly bond near the center of the polypeptide chain (1, 2). Cleavage occurs following treatments that damage DNA or inhibit replication (1-3). Inactivation of LexA is rapid and results in derepression of a set of genes, the SOS regulon, largely responsible for DNA repair. Cleavage of the X repressor is much slower than that of LexA, and more severe treatments are required to induce the prophage. Repressor cleavage in vivo requires RecA function; RecA protein is quiescent during normal cell growth but is activated by the inducing treatment to a form that participates in cleavage.Early in vitro studies showed that the role of RecA protein in cleavage is relatively direct (4). At physiological pH, cleavage is dependent on the presence of RecA protein and two cofactors-a nucleoside triphosphate and single-stranded DNA-that activate RecA by forming a ternary complex. For a time, it was tacitly assumed that activated RecA was a conventional protease with a highly specific active site. This view was challenged by the finding (5) that, under different conditions, cleavage of LexA and the X repressor proceeds spontaneously at the same Ala-Gly bond in an intramolecular reaction, termed "autodigestion," that is stimulated by alkaline pH and does not require RecA. Both RecA-dependent cleavage and autodigestion of LexA are inhibited in a mutant protein, LexA3, that also cannot be cleaved in vivo.It was, therefore, proposed that RecA protein is not itself a protease, but rather that it acts indirectly by stimulating the autodigestion reaction. According to this hypothesis, activated RecA is a positive ef...

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.

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General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.

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