Synthesis and Evaluation of Chloramphenicol Homodimers: Molecular Target, Antimicrobial Activity, and Toxicity against Human Cells
Fig 6
Kinetic analysis of the CAM acetyltransferase reaction using CAM or compounds 4 and 5 as substrates.
The reaction was carried out in 3 ml of 94 mM Tris/HCl pH 7.8, containing 0.083 mM 5,5’-dithio-bis(2-nitrobenzoic acid), 0.16 mM acetyl coenzyme A, 25 units CAM acetyltransferase, and either CAM (●), compound 4 (▲), or compound 5 (■) at the concentrations indicated. The product of the enzymatic reaction, coenzyme A, reacted with 5,5’-dithio-bis(2-nitrobenzoic acid) to yield 5-thio-2-nitrobenzoate which absorbs at 412 nm, with a micromolar extinction coefficient equal to 0.0136. The Vmax and Km values were determined by fitting the substrate concentrations [S] and the obtained ΔA412nm/min (Vo) values into equation V0 = Vmax[S]/(Km + [S]). The obtained Vmax values were divided by 0.0136 to convert their units in μM·min-1 (http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-chloramphenicol-acetyltransferase.html). The ratio Vmax/Km for each curve is given in parenthesis.