title: Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE.
abstract: The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06511, United States.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Dunican
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06511, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Hiller
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06511, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; C465566:RelE protein, E coli