ABSTRACT
Acetyl coenzyme A (acetyl-CoA) carboxylase isozyme 1 (ACC1) and acetyl-CoA carboxylase isozyme 2 (ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation. Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes, and dyslipidemia. One of the major obstacles in the field is the lack of readily-available source of recombinant human ACC enzymes to support systematic drug discovery efforts. Here, we describe an efficient and optimal protocol for expressing and isolating recombinant mammalian ACCs with high yield and purity. The resultant human ACC2, human ACC1, and rat ACC2 possess high specific activities, are properly biotinylated, and exhibit kinetic parameters very similar to the native ACC enzymes. We believe that the current study paves a road to a systematic approach for drug design revolving around the ACC inhibition mechanism.
Subject(s)
Acetyl-CoA Carboxylase/biosynthesis , Acetyl-CoA Carboxylase/isolation & purification , Isoenzymes/biosynthesis , Isoenzymes/isolation & purification , Acetyl-CoA Carboxylase/antagonists & inhibitors , Acetyl-CoA Carboxylase/metabolism , Animals , Baculoviridae , Chromatography, Affinity , Citric Acid/pharmacology , Cloning, Molecular/methods , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Malonyl Coenzyme A/pharmacology , Rats , Recombinant Proteins/isolation & purification , Streptavidin/chemistryABSTRACT
Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.
