Abstract
NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80, 000 by gel filtration and that of the subunit was estimated to be 40, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates.
The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5, 5'-dithiobis (2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.
