The Positively Charged Active Site of the Bacterial Toxin RelE Causes a Large Shift in the General Base pKa
| Autor: | Nan-Sheng Li, Joseph A. Piccirilli, David A. Hiller, Sunitha Nallur, Scott A. Strobel, Brian F. Dunican |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Stereochemistry Bacterial Toxins Mutant Cleavage (embryo) Biochemistry Ribosome Article 03 medical and health sciences Catalytic Domain Side chain RNA Messenger chemistry.chemical_classification 0303 health sciences Messenger RNA biology 030302 biochemistry & molecular biology Active site Hydrogen-Ion Concentration Kinetics A-site Enzyme chemistry Mutation Biocatalysis biology.protein |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b01047 |
| Popis: | The bacterial toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion. |
| Databáze: | OpenAIRE |
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