Kinetic mechanism of NADH‐enoyl‐ACP reductase from Brassica napus

Enoyl-ACP reductase, a component of fatty acid synthase, is a target for anti-microbial agents and herbicides. Here we demonstrate the kinetic mechanism to be a compulsory-order ternary complex with NADH binding before the acyl substrate. Matrix-assisted laser desorption ionisation mass spectrometry analysis of enzymatically and synthesised crotonyl-ACP substrate showed the former to contain a single acyl group, whereas the latter contained up to four additional crotonylations. The use of authentic crotonyl-ACP will be important in future kinetic and crystallographic studies.