Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism

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.

Structure of chloramphenicol acetyltransferase at 1.75-A resolution.

“…All known variants have similar subunit molecular weights (Mr 25,000) and are highly homologous, indicating similar tertiary structure. This conclusion had been inferred from earlier studies of hybrids formed in vivo and in vitro between naturally occurring variants (12, 13 (15), and the type III variant, which has been studied by kinetic and chemical methods (16,17) and which is currently the only variant to yield crystals suitable for x-ray diffraction studies (18).We report here the three-dimensional structures of two binary complexes of CAT, one with the substrate chloramphenicol bound and the second with bound CoA (a product of the forward reaction and substrate for the reverse reaction). Structure Determination Crystals of the binary complex of the enzyme with bound chloramphenicol were obtained by microdialysis ofprotein (5 mg/ml) in 10 mM Mes, pH 6.3, against 2% (vol/vol) 2-methyl-2,4-pentandiol/10 mM Mes, pH 6.3/1 mM chloramphenicol/0.5 mM hexaminecobalt(III) chloride (18).…”

“…All known variants have similar subunit molecular weights (Mr 25,000) and are highly homologous, indicating similar tertiary structure. This conclusion had been inferred from earlier studies of hybrids formed in vivo and in vitro between naturally occurring variants (12, 13 (15), and the type III variant, which has been studied by kinetic and chemical methods (16,17) and which is currently the only variant to yield crystals suitable for x-ray diffraction studies (18).…”

Palmitoylation of bovine opsin and its cysteine mutants in COS cells.

“…4 (25). Similarly, the transfer of acetyl groups to chloramphenicol by chloramphenicol acetyltransferase takes place by a sequential mechanism (26,27).…”

Acyltransferases in Bacteria

“…4]). Thus, an oxyanion tetrahedral intermediate is formed, and the reaction does not follow the alternative, so-called ping-pong mechanism (182).…”