Studies on the Mechanism of Fatty Acid Synthesis

Preparations of enoyl acyl carrier protein (ACP) reductase have been purified 250-fold over crude extracts of Escherichia coli. Such preparations catalyze the reduction of α,β-unsaturated acyl-ACP to saturated derivatives. They utilize both TPNH and DPNH as electron donors for the reduction of the substrate. Available evidence indicates that the TPNH-dependent and the DPNH-dependent reductase activities are due to two distinct enzymes, a TPNH enoyl-ACP reductase and a DPNH enoyl-ACP reductase. The TPNH enoyl reductase is unstable at higher pH values (g7.50) and is enzymatically inactive above pH 8.0. It exhibits an absolute specificity for the acyl-ACP substrates and is more active on crotonyl-ACP than on longer chain α,β-unsaturated acyl-ACP. It has a functional thiol group and can be readily inhibited by p-hydroxymercuribenzoate, iodoacetate, and N-ethylmaleimide. In contrast, the DPNH enoyl-ACP reductase is relatively stable at higher pH values and is enzymatically active over a wide range of pH values. It utilizes both acyl-ACP and acyl coenzyme A substrates and exhibits higher activity with decenoyl-ACP than with crotonyl-ACP. It has a functional thiol group and can be readily inhibited by p-hydroxymercuribenzoate and iodoacetate, but not by N-ethylmaleimide which causes 2- to 3-fold stimulation of enzymatic activity. The reductase reaction is essentially irreversible, and the stoichiometry of the reaction is consistent with the formulation that 1 mole of reduced pyridine nucleotide is consumed for every mole of α,β-unsaturated acyl-ACP reduced. Evidence is presented that the fatty acid-synthesizing system of E. coli requires both TPNH and DPNH for maximum activity. The requirement for DPNH is at the reductase step, and both nucleotides have a synergistic effect in total fatty acid synthesis.