3-(Bromoacetyl)chloramphenicol, an active site-directed inhibitor for chloramphenicol acetyltransferase

Abstract

Bacterial resistance to the antibiotic chloramphenicol is normally mediated by chloramphenicol acetyltransferase (CAT), which utilizes acetyl coenzyme A as the acyl donor in the inactivation reaction. 3-(Bromoacetyl)chloramphenicol, an analogue of the acetylated product of the forward reaction catalyzed by CAT, was synthesized as a probe for accessible and reactive nucleophilic groups within the active site. Extremely potent covalent inhibition was observed. Affinity labeling was demonstrated by the protection afforded by chloramphenicol at concentrations approaching Km for the substrate. Inactivation was stoichiometric, 1 mol of the inhibitor covalently bound per mole of enzyme monomer, with complete loss of both the acetylation and hydrolytic activities associated with CAT. N3-(Carboxymethyl)histidine was identified as the only alkylated amino acid, implicating the presence of a unique tautomeric form of a reactive imidazole group at the catalytic center. The proteolytic digestion of CAT modified with 3-(bromo[14C]-acetyl)chloramphenicol yielded three labeled peptide fractions separable by reverse-phase high-pressure liquid chromatography. Each peptide fraction was sequenced by fast atom bombardment mass spectrometry; the labeled peptide in each case was found to span the highly conserved region in the primary structure of CAT, which had been tentatively assigned as the active site. The rapid, stoichiometric, and specific alkylation of His-189, taken together with the high degree of conservation of the adjacent amino acid residues, strongly suggests a central role for His-189 in the catalytic mechanism of CAT.