Mikrobiologie, Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany.
Anal Biochem. 2011 Apr 1;411(1):100-5. doi: 10.1016/j.ab.2010.11.046. Epub 2010 Dec 5.
Acetyl-coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl-CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl-CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl-CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl-CoA, which is catalyzed by recombinant malonyl-CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening.
乙酰辅酶 A（CoA）羧化酶催化细菌和真核生物脂肪酸生物合成的第一步。该酶是药物设计的靶点，用于治疗人类代谢疾病，以及专门针对草中真核形式酶的除草剂。在药物和除草剂设计中，乙酰辅酶 A 羧化酶活性筛选主要依赖于耗时的酶测定，该测定基于将放射性标记的碳酸氢盐掺入产物丙二酰辅酶 A 中。在这里，我们描述了一种新的简单、连续和快速的比色测定法，避免了放射性底物。它将乙酰辅酶 A 的羧化作用与烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性的丙二酰辅酶 A 还原作用偶联起来，该反应由 Chloroflexus aurantiacus 的重组丙二酰辅酶 A 还原酶催化。该测定法可适用于高通量筛选。
