Abstract
The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.
| Original language | English |
|---|---|
| Pages (from-to) | 211-20 |
| Number of pages | 10 |
| Journal | Biochemical journal |
| Volume | 223 |
| Issue number | 1 |
| Publication status | Published - 1984 |
Keywords
- Acetyl Coenzyme A
- Acetylation
- Acetyltransferases
- Chloramphenicol
- Chloramphenicol O-Acetyltransferase
- Coenzyme A
- Kinetics
- Macromolecular Substances
- Models, Chemical