Enzymology of cottonseed microsomal N-acylphosphatidylethanolamine synthase:: Kinetic properties and mechanism-based inactivation

An ATP-, Ca2+-, and CoA-independent acyltransferase activity, designated "N-acylphosphatidylethanolamine (NAPE) synthase", was reported to catalyze the direct acylation of phosphatidylethanolamine (PE) with free fatty acids (FFAs) in cottonseed microsomes [K.D. Chapman, T.S. Moore, Jr., Plant Physiol. 102(3)(1993) 761-769]. Here, NAPE synthase was purified 138, 176-fold from crude cottonseed homogenates to a specific activity of 5.98 mu mol min(-1) mg(-1) protein by immobilized artificial membrane chromatography. Enzyme purity was confirmed by the presence of a 64 kDa polypeptide in fractions analyzed by tricine-SDS-PAGE. Initial velocity measurements with various free fatty acids ([C-14]-linoleic, -palmitic, -oleic, -stearic and -myristic acids) and saturating concentrations of dioleoyl-PE revealed non-Michaelis-Menten, biphasic kinetics with high and low affinity sites demonstrating positive cooperativity specific for each [C-14]-FFA. In contrast to FFA substrates, no kinetic differences were observed for two different molecular species of PE, (18:1, 18:1)-PE and (16:0, 18:2)-PE, and biphasic curves were not pronounced. Neither [C-14]-dipalmitoylphosphatidylcholine nor [C-14]-palmitoyl-CoA served as acyl donors for the synthesis of NAPE, indicating a preference for FFAs as the acyl donor. Also, neither ethanolamine nor sphingosine functioned as acyl acceptor molecule to form N-acylethanolamine or ceramide, respectively, indicating specificity for the phospholipid PE. NAPE synthase was inactivated in a time-and concentration-dependent manner by diisopropylfluorophosphate (DFP) through the apparent modification of one serine residue. Palmitic acid protected the enzyme from DFP-inactivation and [C-14]-DFP incorporation, suggesting that a serine residue probably binds FFAs in the enzyme's active site forming an acyl-enzyme intermediate. Collectively, these results provide new information on the kinetic behavior of a purified, integral membrane enzyme which synthesizes a bilayer-stabilizing product from two lipid-soluble substrates. The biochemical properties of cottonseed NAPE synthase are consistent with a possible free fatty acid scavenging role in vivo. (C) 1998 Elsevier Science B.V.