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: Brian F Dunican, David A Hiller, Scott A Strobel
• Journal: Biochemistry (Biochemistry) Vol. 54 Issue 47 Pg. 7048-57 (Dec 01 2015) ISSN: 1520-4995 [Electronic] United States
• PMID: 26535789 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Bacterial Toxins
• Escherichia coli Proteins
• Phosphorothioate Oligonucleotides
• RNA, Messenger
• RelE protein, E coli

Topics

• Bacterial Toxins (chemistry, genetics, metabolism)
• Catalytic Domain
• Escherichia coli (chemistry, genetics, metabolism)
• Escherichia coli Proteins (chemistry, genetics, metabolism)
• Hydrogen-Ion Concentration
• Kinetics
• Models, Molecular
• Mutation
• Phosphorothioate Oligonucleotides (chemistry, metabolism)
• RNA, Messenger (chemistry, metabolism)