A study of the enzymatic inactivation of chloramphenicol by highly purified chloramphenicol acetyltransferase.
We report the purification of chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) by a two-step procecdure involving chromatography on a Sepharose 4B-reduced chloramphenicol matrix and DEAE-Sephadex A-50. This procedure resulted in a 120-fold purification with 50% recovery of the enzyme. Only one band of enzyme activity was present after electrophoresis on polyacrylamide gel. The enzyme is active over a broad pH range, maximal activity being observed near pH 7.6. Both chloramphenicol 1-acetate of chloramphenicol 3-acetate were found to be very stable in Tris-maleate buffer at pH 6.09 with negligible interconversion. The incubation at pH 6.0 of chloramphenicol 1-acetate with the purified chloramphenicol acetyltransferase yielded chloramphenicol 1,3-diacetate. These data indicate that the enzyme acetylates specifically at the 3-hydroxy position and the diacetylation is possible only because of non-enzymatic interconversion of chloramphenicol 3-acetate to chloramphenicol 1-acetate at higher pH values.
D000107:Acetylation; D000123:Acetyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D002701:Chloramphenicol / Q000031:analogs & derivatives* / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity
C026822:chloramphenicol 3-acetate; C026821:chloramphenicol 1-acetate; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase