Spectrophotometric assay of 2,4-dienoyl coenzyme a reductase with 5-phenyl-2,4-pentadienoyl-coenzyme a as substrate

The spectrophotometric assay of 2,4-dienoyl coenzyme A (CoA) reductase (EC 1.1.1.34) was modified to improve the linearity and sensitivity of this method. 5-Phenyl-2,4-pentadienoyl-CoA, which has an absorbance maximum at 340 nm with an extinction coefficient of 44,300 M⁻¹ cm⁻¹, was synthesized and used as substrate. This compound is reduced by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent 2,4-dienoyl-CoA reductase to 5-phenyl-3-pentenoyl-CoA. When a tissue homogenate serves as an enzyme source, the product is further metabolized by Δ³Δ²-enoyl-CoA isomerase (EC 5.3.3.8) to 5-phenyl-2-pentenoyl-CoA, which is hydrated to 5-phenyl-3-hydroxypentanoyl-CoA by enoyl-CoA hydratase (EC 4.2.1.17). The modified assay method, which measures the decrease in absorbance at 340 nm due to the reduction of 5-phenyl-2,4-pentadienoyl-CoA and the oxidation of NADPH, is linear for a longer period of time and is twice as sensitive as the conventional assay with 2,4-decadienoyl-CoA as substrate.