Assessing Acetyl-Coenzyme A Carboxylase Activity and Inhibition in Using a Partially Purified Protein Extract.

Acetyl-coenzyme A carboxylase (ACC) catalyzes the ATP-dependent carboxylation of acetyl-CoA into malonyl-CoA, the first step in fatty acid biosynthesis. ACC is increasingly recognized as being crucial for energy metabolism, leading to its emergence as a potential therapeutic target mainly for obesity and cancer. All previous ACC kinetics studies were conducted with pure enzyme preparations and measurements at several substrate concentrations using methods such as radioactivity counting of labeled substrate(s) and/or product(s) or UV estimation of chromatographically resolved components. In alternative, a real-time kinetics method based on HPLC or NMR monitoring was developed and successfully applied using partially purified protein extracts and a single substrate concentration. Kinetic parameters were derived for ACC in the absence and presence of the ACC inhibitor avocadene acetate, a compound present in avocadoes. Both monitoring techniques yielded consistent kinetic parameters that compared well with previously reported values and provided new insights into the inhibition mechanism.