| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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The Δ3-Δ2-Enoyl-CoA isomerase and 2-enoyl-CoA hydratase-1 activity measurements were performed as described by Palosaari and Hiltunen (1990) using 60 µM trans-3-hexenoyl-CoA and crotonyl-CoA as substrates, respectively. The reaction was monitored by following the generation of a magnesium complex of 3-ketoacyl-CoA at 303 nm using a Shimadzu UV 3000 spectrophotometer. The substrates were synthesized by Anna-Leena Hietajärvi and Werner Schmitz using the mixed anhydride method (Rasmussen et al. 1990).
For the determination of Δ3,5-Δ2,4-dienoyl-CoA isomerase enzyme activity, the substrate, 3,5,8,11,14-eicosapentenoyl-CoA, was generated by incubating 60 µM arachidonoyl-CoA (5,8,11,14-eicosapentenoyl-CoA) (Sigma) with 0.2 U yeast acyl-CoA oxidase (Sigma) as described earlier (Filppula et al. 1998). The formation of the Δ2-Δ4 conjugated double bond caused by Δ3,5-Δ2,4-dienoyl-CoA isomerase activity was detected spectrophotometrically at 300 nm.
To determine kinetic parameters, 5, 10, 20, 40, 60, 100 and 200 µM of trans-3-hexenoyl-CoA were used, and the Km and Vmax values were calculated using GraFit software (Sigma).