Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.
Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.
D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002701:Chloramphenicol; D002846:Chromatography, Affinity; D004587:Electrophoresis, Agar Gel; D007700:Kinetics; D010777:Photochemistry; D013210:Staphylococcus / Q000201:enzymology*
D000596:Amino Acids; D002701:Chloramphenicol; D000123:Acetyltransferases