Studies on a chloramphenicol acetyltransferase variant.

An E.coli Chloramphenicol Acetyltransferase Type III variant has been purified and characterized. The variant has been shown to be related to, but not identical, to the E.coli Type I and Type II variants. Inhibition studies with the reagents iodoacetamide and iodoacetate revealed that iodoacetamide was 15 times as effective as an inhibitor as was iodoacetate. Experiments with [14C] iodoacetamide gave an incorporation of approximately 1 mole of [14C] label per mole of enzyme monomer. Two unique radioactive peptides were isolated and sequence analysis indicated that a histidine and a cysteine residue were modified. The substrates, chloramphenicol and acetyl-S-CoA were both able to protect against the loss of activity. To test the hypothesis that a negatively charged residue was near the active site of the variant the 5,5 dithiobis (2-nitrobenzoic acid) analogues, 5,5 dithiobis (2-nitromethylbenzoate) and 5,5 dithio-bis (2-nitrotoluene) were characterized. The latter was found not to be suitable as an inhibitor but the former rapidly inhibited the enzyme. Chloramphenicol was able to protect against the loss of activity, supporting the hypothesis that a reactive sulphydryl group existed near or in the chloramphenicol binding site. The histidine residue was further investigated using the reagents diethylpyrocarbonate (DEP) and methyl nitrobenzene sulphonate (MNBS). DEP modified two histidine residues, one within the chloramphenicol binding site and one outside the substrate binding sites. Radioactively labelled MNBS was synthesised and analysis of the modified enzyme revealed the formation of 3-methylhistidine. The acetyl-S-CoA analogues, acetonyl-S-CoA and methyl-S-CoA, were synthesised and characterized. Methyl-S-CoA was found to accelerate the rate of inhibition seen with MNBS and increased the dissociation constant of chloramphenicol with respect to the enzyme. Preliminary studies were also carried out with the reagents rose bengal, butanedione and an epoxide antibiotic. The data suggests that a reactive histidine and cysteine residue exists within or near the active site of the variant. A proposed reaction mechanism with the histidine residue acting as a general base is postulated