Intramolecular cleavage of lexA and phage .lambda. repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent

Abstract

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.

Mutagenic DNA repair in enterobacteria

“…After exposure to DNA replicationinhibiting agents such as UV light, the autocatalytic cleavage of LexA results in the inactivation of repressor function and leads to derepression of SOS-controlled genes. In vitro this reaction occurs at alkaline pH or, at more physiological conditions, after interaction with a ternary complex of RecA protein, single-stranded DNA, and ATP (19, 42,43). Elevated levels of the UmuDC proteins are not sufficient to promote mutagenesis.…”

Mutant LexA proteins with an increased rate of in vivo cleavage.

“…This regulon is controlled primarily by the action of LexA LexA cleavage also occurs in vitro under two different conditions: (i) RecA-mediated cleavage can occur at neutral pH and requires RecA and two types of cofactors that activate the RecA (3); (ii) at alkaline pH an intramolecular and RecA-independent cleavage, termed autodigestion, takes place (4)(5)(6). Both reactions cleave a specific peptide bond between Ala-84 and Gly-85 of LexA (202 amino acids in size), and both are impaired by many mutations, termed lexA (Ind-), which we will refer to here as Ind-, that were isolated on the basis of defects in in vivo RecA-mediated cleavage (4,7,8).…”

“…Although autodistion of LexA is an intramolecular reaction with all the reactants present in high local concentrations, its maximalrate is rather slow, with a half-life in vitro of 5 (17); 806 plates contained 2.0o Difco agar, 0.4% glucose, 0.02% arginine, 0.01% histidine, 0.01% isoleucine, 0.01% valine, 0.01% threonine, 0.01% leucine, 0.007% methionine and thiamine (2 (8). pJWL59 carries a fusion of lexA' to the tac promoter (5).…”

“…pJWL59 carries a fusion of lexA' to the tac promoter (5). Position 92 alleles were moved into pJWL184 by subcloning with BamHI and Sal I; tac promoter versions were made by subcloning the promoter-containing EcoRI-Mlu I .fragment of pJWL59.…”

“…In oligonucleotide 2 (5'-CTG GCG CAA *** CAT ATT GAA GG-3'), the three sites shown with asterisks were synthesized by using a mixture of 33% of the nucleotides 5 (16).…”